Acyclic nucleoside derivatives

ABSTRACT

Methods and novel intermediates for the preparation of and the treatment with acyclic nucleoside derivatives of the formula: 
     
       
         
         
             
             
         
       
         
         
           
             where one of R 1  and R 2  is an amino acid acyl group and the other of R 1  and R 2  is a —C(O)C 3 -C 21  saturated or monounsaturated, optionally substituted alkyl and 
             R 3  is OH or H.

This application is a Divisional of co-pending application Ser. No.10/741,615, filed on Dec. 19, 2003, which is a Divisional of applicationSer. No. 10/076,833, filed on Feb. 14, 2002 now U.S. Pat. No. 6,703,394,which is a Divisional of application Ser. No. 09/550,554, filed on Apr.17, 2000 now U.S. Pat. No. 6,576,763, which is a Divisional ofapplication Ser. No. 09/146,194, which was filed on Sep. 3, 1998 nowU.S. Pat. No. 6,225,312, which is a Divisional of application Ser. No.08/798,216, which was filed on Feb. 10, 1997 now U.S. Pat. No.5,869,493, the entire contents of +each of which are hereby incorporatedby reference and for which priority is claimed under 35 U.S.C. §120.This application also claims priority of Application Nos. 9600613-5 and9600614-3, both filed in Sweden on Feb. 16, 1996 under 35 U.S.C. §119.

TECHNICAL FIELD

This invention relates to the field of antivirals and in particular toderivatives of acyclic nucleosides useful against herpes and retroviralinfections. The invention provides novel compounds, pharmaceuticalcompositions comprising these compounds, methods for the treatment orprophylaxis of viral infections employing them, methods for theirmanufacture and novel intermediates.

BACKGROUND TO THE INVENTION

The practical utility of many acyclic nucleosides is limited by theirrelatively modest pharmacokinetics. A number of prodrug approaches havebeen explored in an effort to improve the bioavailability of acyclicnucleosides in general. One of these approaches involves the preparationof ester derivatives, particularly aliphatic esters, of one or more ofthe hydroxy groups on the acyclic side chain.

European patent EP 165 289 describes the promising antiherpes agent9-[4-hydroxy-(2-hydroxymethyl)butyl]guanine, otherwise known as H2G.European patent EP 186 640 discloses 6-deoxy H2G. European patent EP 343133 discloses that these compounds, particularly the R-(−) enantiomer,are additionally active against retroviral infections such as HIV.Various derivatives of H2G, such as phosphonates, aliphatic esters (forexample, the diacetate and the dipropionate) and ethers of the hydroxygroups on the acyclic side chain are disclosed in EP 343 133. Thispatent also discloses methods for the preparation of these derivativescomprising the condensation of the acyclic side chain to the N-9position of a typically 6-halogenated purine moiety or, alternatively,the imidazole ring closure of a pyrimidine or furazano-[3,4-d]pyrimidinemoeity or the pyrimidine ring closure of an imidazole moiety, where theacyclic side chain is already present in the precursor pyrimidine orimidazole moiety, respectively. In the broadest description of each ofthese methods the acyclic side chain is pre-derivatised but individualexamples also show a one-step diacylation of H2G with acetic orproprionic anhydride and DMF.

Harnden, et al., J. Med. Chem. 32, 1738 (1989) investigated a number ofshort chain aliphatic esters of the acyclic nucleoside9-[4-hydroxy-(3-hydroxymethyl)butyl]guanine, otherwise known aspenciclovir, and its 6-deoxy analog. Famciclovir, a marketed antiviralagent, is the diacetyl derivative of 6-deoxy penciclovir.

Benjamin, et al., Pharm. Res. 4 No. 2, 120 (1987) discloses short chainaliphatic esters of 9-[(1,3-dihydroxy-2-propoxy)-methyl]guanine,otherwise known as ganciclovir. The dipropionate ester is disclosed tobe the preferred ester.

Lake-Bakaar, et al., discloses in Antimicrob. Agents Chemother. 33 No.1, 110-112 (1989) diacetate and dipropionate derivatives of H2G andmonoacetate and diacetate derivatives of 6-deoxy H2G. The diacetate anddipropionate derivatives of H2G are reported to result in only modestimprovements in bioavailability relative to H2G.

International patent application WO94/24134, published Oct. 27, 1994,discloses aliphatic ester prodrugs of the 6-deoxy N-7 analog ofganciclovir, including the di-pivaloyl, di-valeroyl, mono-valeroyl,mono-oleoyl and mono-stearoyl esters.

International patent application WO93/07163, published Apr. 15, 1993 andInternational patent application WO94/22887, published Oct. 13, 1994,both disclose mono-ester derivatives of nucleoside analogs derived frommono-unsaturated C18 or C20 fatty acids. U.S. Pat. No. 5,216,142, issuedJun. 1, 1993, also discloses long chain fatty acid mono-esterderivatives of nucleoside analogs.

A second approach to providing prodrugs of acyclic nucleosides involvesthe preparation of amino acid esters of one or more of the hydroxygroups on the acyclic side chain. European patent EP 99 493 disclosesgenerally amino acid esters of acyclovir and European patent applicationEP 308 065, published Mar. 22, 1989, discloses the valine and isoleucineesters of acyclovir.

European patent application EP 375 329, published Jun. 27, 1990,discloses amino acid ester derivatives of ganciclovir, including thedi-valine, di-isoleucine, di-glycine and di-alanine ester derivatives.International patent application WO95/09855, published Apr. 13, 1995,discloses amino acid ester derivatives of penciclovir, including themono-valine and di-valine ester derivatives.

DE 19526163, published Feb. 1, 1996 and U.S. Pat. No. 5,543,414 issuedAug. 6, 1996, disclose achiral amino acid esters of ganciclovir.

European patent application EP 694 547, published Jan. 31, 1996,discloses the mono-L-valine ester of ganciclovir and its preparationfrom di-valyl-ganciclovir.

European patent application EP 654 473, published May 24, 1995,discloses various his amino acid ester derivatives of9-[1′,2′-bishydroxymethyl)-cyclopropan-1′yl]methylguanine.

International patent application WO95/22330, published Aug. 24, 1995,discloses aliphatic esters, amino acid esters and mixed acetate/valinateesters of the acyclic nucleoside9-[3,3-dihydroxymethyl-4-hydroxy-but-1-yl]guanine. This referencediscloses that bioavailability is reduced when one of the valine estersof the trivaline ester derivative is replaced with an acetate ester.

BRIEF DESCRIPTION OF THE INVENTION

We have found that diester derivatives of H2G bearing specificcombinations of an amino acid ester and a fatty acid ester are able toprovide significantly improved oral bioavailability relative to theparent compound (H2G). In accordance with a first aspect of theinvention there is thus provided novel compounds of the formula I

-   -   where a) R₁ is —C(O)CH(CH(CH₃)₂)NH₂ or —C(O)CH(CH(CH₃)CH₂CH₃)NH₂        and R₂ is —C(O)C₃-C₂₁ saturated or monounsaturated, optionally        substituted alkyl; or        -   b) R₁ is —C(O)C₃-C₂₁ saturated or monounsaturated,            optionally substituted alkyl and R₂ is —C(O)CH(CH(CH₃)₂)NH₂            or —C(O)CH(CH(CH₃)CH₂CH₃)NH₂; and    -   R₃ is OH or H;    -   and pharmaceutically acceptable salts thereof.

The advantageous effect on oral bioavailability of the mixed fatty acidand amino acid esters of the invention is particularly unexpected incomparison to the oral bioavailability of the corresponding fatty acidesters. Based on the results using a urinary recovery assay (Table 1A)or a plasma drug assay (Table 1B) of H2G from rats, neither the mono ordi-fatty acid esters of H2G provide any improvement in oralbioavailability relative to the parent compound H2G. Indeed thedi-stearate derivative provided significantly lower bioavailability thanthe parent indicating that a stearate ester may be detrimental forimproving oral bioavailability of H2G. Converting one or both of thehydroxyls in certain other acyclic nucleoside analogues to thecorresponding valine or di-valine ester has been reported to improvebioavailability. Conversion of H2G to the corresponding mono- ordi-valyl ester derivatives produced similar improvement inbioavailability relative to the parent compound. Given that fatty acidderivatives of H2G are shown to be detrimental for improvingbioavailability, it was unexpected that a mixed amino acid/fatty aciddiester derivative of H2G would provide improved or comparable oralbioavailability to that of the valine diester derivative of H2G, basedon urine recovery and plasma drug assays, respectively.

TABLE 1A R₁ group R₂ group Bioavailability* hydrogen hydrogen 8%hydrogen stearoyl 12% stearoyl stearoyl 1% valyl hydrogen 29% valylvalyl 36% valyl stearoyl 56% *see Biological Example 1 below for details

TABLE 1B R₁ group R₂ group Bioavailability^(#) hydrogen hydrogen 3.8%hydrogen stearoyl 1.9% stearoyl stearoyl   0% valyl hydrogen 31.3% valyl valyl 35.0%  valyl stearoyl  29% ^(#)see Biological Example 2below for details

The invention also provides pharmaceutical compositions comprising thecompounds of Formula I and their pharmaceutically acceptable salts inconjunction with a pharmaceutically acceptable carrier or diluent.Further aspects of the invention include the compounds of Formula I andtheir pharmaceutically acceptable salts for use in therapy and the useof these compounds and salts in the preparation of a medicament for thetreatment or prophylaxis of viral infection in humans or animals.

The compounds of the invention are potent antivirals, especially againstherpes infections, such as those caused by Varicella zoster virus,Herpes simplex virus types 1 & 2, Epstein-Barr virus, Herpes type 6(HHV-6) and type 8 (HHV-8). The compounds are particularly usefulagainst Varicella zoster virus infections such as shingles in theelderly including post herpetic neuralgia or chicken pox in the youngwhere the duration and severity of the disease can be reduced by severaldays. Epstein Barr virus infections amenable to treatment with thecompounds include infectious mononucleosis/glandular fever which haspreviously not been treatable but which can cause many months ofscholastic incapacity amongst adolescents.

The compounds of the invention are also active against certainretroviral infections, notably SIV, HIV- and HIV-2, and againstinfections where a transactivating virus is indicated.

Accordingly a further aspect of the invention provides a method for theprophylaxis or treatment of a viral infection in humans or animalscomprising the administration of an effective amount of a compound ofFormula I or its pharmaceutically acceptable salt to the human oranimal.

Advantageously group R₃ is hydroxy or its tautomer ═O so that the baseportion of the compounds of the invention is the naturally occurringguanine, for instance in the event that the side chain is cleaved invivo. Alternatively, R₃ may be hydrogen thus defining the generally moresoluble 6-deoxy derivative which can be oxidised in vivo (e.g. byxanthine oxidase) to the guanine form.

The compound of formula I may be present in racemic form, that is amixture of the 2R and 2S isomers. Preferably, however, the compound offormula I has at least 70%, preferably at least 90% R form, for examplegreater than 95%. Most preferably the compound of formula I isenantiomerically pure R form.

Preferably the amino acid of group R₁/R₂ is derived from an L-aminoacid.

Preferably the fatty acid of group R₁/R₂ has in total an even number ofcarbon atoms, in particular, decanoyl (C₁₀), lauryl (C₁₂), myristoyl(C₁₄), palmitoyl (C₁₆), stearoyl (C₁₈) or eicosanoyl (C₂₀). Other usefulR₁/R₂ groups include butyryl, hexanoyl, octanoyl or behenoyl (C₂₂).Further useful R₁/R₂ groups include those derived from myristoleic,myristelaidic, palmitoleic, palmitelaidic, n6-octadecenoic, oleic,elaidic, gandoic, erucic or brassidic acids. Monounsaturated fatty acidesters typically have the double bond in the trans configuration,preferably in the ω-6, ω-9 or ω-11 position, dependent upon theirlength. Preferably the R₁/R₂ group is derived from a fatty acid whichcomprises a C₉ to C₁₇ saturated, or n:9 monounsaturated, alkyl.

The saturated or unsaturated fatty acid or R₁/R₂ may optionally besubstituted with up to five similar or different substituentsindependently selected from the group consisting of such as hydroxy,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkoxy C₁-C₆ alkyl, C₁-C₆ alkanoyl,amino, halo, cyano, azido, oxo, mercapto and nitro, and the like.

Most preferred compounds of the formula I are those where R₁ is—C(O)CH(CH₃)₂)NH₂ or —C(O)CH(CH(CH₃)CH₂CH₃)NH₂ and R₂ is —C(O)C₉-C₁₇saturated alkyl.

The term “lower alkyl” as used herein refers to straight or branchedchain alkyl radicals containing from 1 to 7 carbon atoms including, butnot limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2,2-dimethylbutyl,2-methylpentyl, 2,2-dimethylpropyl, n-hexyl and the like.

The term “N-protecting group” or “N-protected” as used herein refers tothose groups intended to protect the N-terminus of an amino acid orpeptide or to protect an amino group against undesirable reactionsduring synthetic procedures. Commonly used N-protecting groups aredisclosed in Greene, “Protective Groups in Organic Synthesis” (JohnWiley & Sons, New York, 1981), which is hereby incorporated byreference. N-protecting groups include acyl groups such as formyl,acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,2-bromoacetyl, trifluoracetyl, trichloroacetyl, phthalyl,o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl,4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such asbenzenesulfonyl, p-toluenesulfonyl, and the like, carbamate forminggroups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl andthe like; and silyl groups such as trimethylsilyl and the like. FavouredN-protecting groups include formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, phenylsulfonyl, benzyl, t-butoxycarbonyl (BOC) andbenzyloxycarbonyl (Cbz).

The term “activated ester derivative” as used herein refers to acidhalides such as acid chlorides, and activated esters including, but notlimited to, formic and acetic acid derived anhydrides, anhydridesderived from alkoxycarbonyl halides such as isobutyloxycarbonylchlorideand the like, N-hydroxysuccinimide derived esters, N-hydroxyphthalimidederived esters, N-hydroxybenzotriazole derived esters,N-hydroxy-5-norbornene-2,3-dicarboxamide derived esters,2,4,5-trichlorophenyl derived esters and the like.

Preferred compounds of formula I include:

-   (R)-9-[2-(butyryloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,-   (R)-9-[2-(4-acetylbutyryloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,-   (R)-9-[2-(hexanoyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,-   (R)-9-[4-(L-isoleucyloxy)-2-(octanoyloxymethyl)butyl]guanine,-   (R)-9-[4-(L-isoleucyloxy)-2-(decanoyloxymethyl)butyl]guanine,-   (R)-9-[4-(L-isoleucyloxy)-2-(dodecanoyloxymethyl)butyl]guanine,-   (R)-9-[4-(L-isoleucyloxy)-2-(tetradecanoyloxymethyl)butyl]guanine,-   (R)-9-[4-(L-isoleucyloxy)-2-(hexadecanoyloxymethyl)butyl]guanine,-   (R)-9-[4-(L-isoleucyloxy)-2-(octadecanoyloxymethyl)butyl]guanine,-   (R)-9-[2-(eicosanoyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,-   (R)-9-[2-(docosanoyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,-   (R)-9-[4-(L-isoleucyloxy)-2-((9-tetradecenoyl)oxymethyl)butyl]guanine,-   (R)-9-[2-((9-hexadecenoyl)oxymethyl)-4-(L-isoleucyloxy)butyl]guanine,-   (R)-9-[4-(L-isoleucyloxy)-2-((6-octadecenoyl)oxymethyl)butyl]guanine,-   (R)-9-[4-(L-isoleucyloxy)-2-((9-octadecenoyl)oxymethyl)-butyl]guanine,-   (R)-9-[2-((11-eicosanoyl)-oxymethyl)-4-(L-isoleucyloxy)butyl]guanine,-   (R)-9-[2-((13-docosenoyl)-oxymethyl)-4-(L-isoleucyloxy)butyl]guanine,-   (R)-2-amino-9-[2-(butyryloxymethyl)-4-(L-isoleucyloxy)butyl]purine,-   R)-2-amino-9-[2-(4-acetylbutyryloxymethyl)-4-(L-isoleucyloxy)butyl]purine,-   (R)-2-amino-9-[2-(hexanoyloxymethyl)-4-(L-isoleucyloxy)butyl]purine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-(octanoyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-(decanoyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-(dodecanoyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-(tetradecanoyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-(hexadecanoyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-(octadecanoyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-(eicosanoyloxymethyl)butyl]purine,-   (R)-2-amino-9-[2-(eicosanoyloxymethyl)-4-(L-isoleucyloxy)butyl]purine,-   (R)-2-amino-9-[2-(docosanoyloxymethyl)-4-(L-isoleucyloxy)butyl]purine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-((9-tetradecenoyl)oxymethyl)butyl]purine,-   (R)-2-amino-9-[2-((9-hexadecenoyl)oxymethyl)-4-(L-isoleucyloxy)butyl]purine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-((6-octadecenoyl)oxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-((9-octadecenoyl)oxymethyl)butyl]purine,-   (R)-2-amino-9-[2-((11′-eicosanoyl)oxymethyl)-4-(L-isoleucyloxy)butyl]purine,    or-   (R)-2-amino-9-[2-((13-docosenoyl)oxymethyl)-4-(L-isoleucyloxy)butyl]purine,    and their pharmaceutically acceptable salts.

Further preferred compounds include:

-   (R)-9-[2-(butyryloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-(4-acetylbutyryloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-(hexanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-(octanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-(decanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-(dodecanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-(tetradecanoyloxymethyl-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-hexadecanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-(octadecanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-(eicosanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-(eicosanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-(docosanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-((9-tetradecenoyl)oxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-((9-hexadecenoyl)oxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-((6-octadecenoyl)oxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-((9-octadecenoyl)oxymethyl)-4-(L-valyloxy)-butyl]guanine,-   (R)-9-[2-((11-eicosanoyl)oxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-((13-docosenoyl)oxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-2-amino-9-[2-(butyryloxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-(4-acetylbutyryloxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-(hexanoyloxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-(octanoyloxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-(decanoyloxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-(dodecanoyloxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-(tetradecanoyloxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-(hexadecanoyloxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-(octadecanoyloxymethyl)-4-(L-valyloxy)-butyl]purine,-   (R)-2-amino-9-[2-(eicosanoyloxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-(docosanoyloxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-((9-tetradecenoyl)oxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-((9-hexadecenoyl)oxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-((6-octadecenoyl)oxymethyl)-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-((9-octadecenoyl)oxymethyl)-4-(L-valyloxy)-butyl]purine,-   (R)-2-amino-9-[2-((11-eicosenoyl)-oxymethyl)-4-(L-valyloxy)butyl]purine,    or-   (R)-2-amino-9-[2-((13-docosenoyl)-oxymethyl)-4-(L-valyloxy)butyl]purine;    and their pharmaceutically acceptable salts.

Other preferred compounds of formula I include:

-   (R)-9-[4-(butyryloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-(4-acetylbutyryloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-(hexanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-(octanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-(decanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-(dodecanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-(tetradecanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-hexadecanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-(octadecanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-(eicosanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-(docosanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-((9-tetradecenoyl)oxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-((9-hexadecenoyl)oxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-((6-octadecenoyl)oxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-((9-octadecenoyl)oxy)-2-(L-valyloxymethyl)-butyl]guanine,-   (R)-9-[4-((11-eicosenoyl)oxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-9-[4-((13-docosenoyl)-oxy)-2-(L-valyloxymethyl)butyl]guanine,-   (R)-2-amino-9-[4-(butyryloxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(4-acetylbutyryloxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(hexanoyloxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(octanoyloxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(decanoyloxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(dodecanoyloxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(tetradecanoyloxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(hexadecanoyloxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(octadecanoyloxy)-2-(L-valyloxymethyl)-butyl]purine,-   (R)-2-amino-9-[4-(eicosanoyloxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-(docosanoyloxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-((9-tetradecenoyl)oxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-((9-hexadecenoyl)oxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-((6-octadecenoyl)oxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-((9-octadecenoyl)oxy)-2-(L-valyloxymethyl)butyl]purine,-   (R)-2-amino-9-[4-((11-eicosenoyl)oxy)-2-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-((13-docosenoyl)oxymethyl)-2-(L-valyloxy)butyl]purine,    or and their pharmaceutically acceptable salts.

The compounds of formula I can form salts which form an additionalaspect of the invention. Appropriate pharmaceutically acceptable saltsof the compounds of formula I include salts of organic acids, especiallycarboxylic acids, including but not limited to acetate,trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate,malate, pantothenate, isethionate, adipate, alginate, aspartate,benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate,glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate,palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate,tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate,organic sulphonic acids such as methanesulphonate, ethanesulphonate,2-hydroxyethane sulphonate, camphorsulphonate, 2-napthalenesulphonate,benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate;and inorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoricand sulphonic acids. Hydrochloric acid salts are convenient.

The compounds of Formula I may be isolated as the hydrate. The compoundsof the invention may be isolated in crystal form, preferably homogenouscrystals, and thus an additional aspect of the invention provides thecompounds of Formula I in substantially pure crystalline form,comprising >70%, preferably >90% homogeneous crystalline material forexample >95% homogeneous crystalline material.

The compounds of the invention are particularly suited to oraladministration, but may also be administered rectally, vaginally,nasally, topically, transdermally or parenterally, for instanceintramuscularly, intravenously or epidurally. The compounds may beadministered alone, for instance in a capsule, but will generally beadministered in conjunction with a pharmaceutically acceptable carrieror diluent. The invention extends to methods for preparing apharmaceutical composition comprising bringing a compound of Formula Ior its pharmaceutically acceptable salt in conjunction or associationwith a pharmaceutically acceptable carrier or vehicle.

Oral formulations are conveniently prepared in unit dosage form, such ascapsules or tablets, employing conventional carriers or binders such asmagnesium stearate, chalk, starch, lactose, wax, gum or gelatin.Liposomes or synthetic or natural polymers such as HPMC or PVP may beused to afford a sustained release formulation. Alternatively theformulation may be presented as a nasal or eye drop, syrup, gel or creamcomprising a solution, suspension, emulsion, oil-in-water orwater-in-oil preparation in conventional vehicles such as water, saline,ethanol, vegetable oil or glycerine, optionally with flavourant and/orpreservative and/or emulsifier.

The compounds of the invention may be administered at a daily dosegenerally in the range 0.1 to 200 mg/kg/day, advantageously, 0.5 to 100mg/kg/day, more preferably 10 to 50 mg/kg/day, such as 10 to 25mg/kg/day. A typical dosage rate for a normal adult will be around 50 to500 mg, for example 300 mg, once or twice per day for herpes infectionsand 2 to 10 times this dosage for HIV infections.

As is prudent in antiviral therapy, the compounds of the invention canbe administered in combination with other antiviral agents, such asacyclovir, valcyclovir, penciclovir, famciclovir, ganciclovir and itsprodrugs, cidofovir, foscarnet and the like for herpes indications andAZT, ddI, ddC, d4T, 3TC, foscarnet, ritonavir, indinavir, saquinavir,delaviridine, Vertex VX 478, Agouron AG1343 and the like for retroviralindications.

The compounds of the invention can be prepared de novo or byesterification of the H2G parent compound which is prepared, forexample, by the synthesis methodology disclosed in European Patent EP343 133, which is incorporated herein by reference.

A typical reaction scheme for the preparation of H2G is depictedoverleaf:

The condensation in step 1 is typically carried out with a base catalystsuch as NaOH or Na₂CO₃ in a solvent such as DMF. Step 2 involves areduction which can be performed with LiBH/tetrahydrofuran in a solventsuch as t-BuOH. The substitution in step 3 of the chlorine with an aminogroup can be performed under pressure with ammonia. Step 4 employsadenosine deaminase which can be conveniently immobilized on a solidsupport. Cooling the reaction mixture allows unreacted isomericprecursor to remain in solution thereby enhancing purity.

Starting materials for compounds of the invention in which R₃ ishydrogen may be prepared as shown in European Patent EP 186 640, thecontents of which are incorporated herein by reference. These startingmaterials may be acylated as described for H2G below, optionally afterprotecting the purine 2-amino group with a conventional N-protectinggroup as defined above, especially BOC (t-BuO—CO—), Z (BnO—CO—) orPh₃C—.

The compounds of the invention may be prepared from H2G as describedbelow in Schemes A and B.

A. Direct Acylation Method

Scheme A depicts the preparation of compounds in which R₁ is derivedfrom the amino acid and R₂ is derived from the fatty acid, but theconverse scheme is applicable to compounds where R₁ is derived from thefatty acid and R₂ is derived from the amino acid ester. In the variantspecifically depicted in scheme A above, G is guanine or 6-deoxyguanine,PG is an optional N-protecting group or hydrogen, R₁* is the valine orisoleucine side chain and R₂* is the fatty acid chain. H2G is depictedabove as a starting material but this of course may be optionallyprotected at R₃ or the 2 position of the purine with conventionalN-protecting groups (not shown). The H2G (derivative) reacts in thefirst step with an activated R₁ α-amino acid derivative, as furtherdescribed below, in a solvent such as dimethylformamide or pyridine, togive a monoacylated product. The R₁ α-amino acid may be suitablyN-protected with N—BOC or N—CBz or the like. Under controlledconditions, the first acylation can be made to predominantly take placeat the side chain 4-hydroxy group on the side chain of H2G. Thesecontrolled conditions can be achieved, for example, by manipulating thereagent concentrations or rate of addition, especially of the acylatingagent, by lowering the temperature or by the choice of solvent. Thereaction can be followed by TLC to monitor the controlled conditions.

After purification, the R₁ monoacylated compounds are further acylatedon the side chain 2-CH₂OH group with the appropriate activated fattyacid derivative to give diacylated products using similar procedures asfor the first esterification step. The diester products are subsequentlysubjected to a conventional deprotection treatment using for exampletrifluoroacetic acid, HCl(aq)/dioxane or hydrogenation in the presenceof catalyst to give the desired compound of Formula I. The compound maybe in salt form depending on the deprotection conditions.

The activated R₁/R₂ acid derivative used in the various acylations maycomprise e.g. the acid halide, acid anhydride, activated acid ester orthe acid in the presence of coupling reagent, for exampledicyclohexylcarbodiimide, where “acid” in each case represents thecorresponding R₁/R₂ amino acid or the R₁/R₂ fatty acid. Representativeactivated acid derivatives include the acid chloride, formic and aceticacid derived mixed anhydrides, anhydrides derived from alkoxycarbonylhalides such as isobutyloxycarbonylchloride and the like,N-hydroxysuccinamide derived esters, N-hydroxyphthalimide derivedesters, N-hydroxy-5-norbornene-2,3-dicarboxamide derived esters,2,4,5-trichlorophenol derived esters and the like.

B. Via Protection of the Side Chain 4-Hydroxy Group:

wherein G, PG, R₁* and R₂* are as described for scheme A.

Scheme B has been exemplified with reference to the preparation of acompound where R₁ is derived from an amino acid and R₂ is derived fromthe fatty acid ester, but a converse scheme will be applicable tocompounds where R₂ is derived from the amino acid and R₁ is derived fromthe fatty acid. This scheme relies on regioselective protection of theH2G side chain 4-hydroxy group with a bulky protecting group. In schemeB above this is depicted as t-butyldiphenylsilyl, but otherregioselective protecting groups such as trityl, 9-(9-phenyl)xanthenyl,1,1-bis(4-methylphenyl)-1′-pyrenylmethyl may also be appropriate. Theresulting product is acylated at the side chain 2-hydroxymethyl groupusing analogous reagents and procedures as described in scheme A above,but wherein the activated acid derivative is the R₂ fatty acid, forexample, myristic, stearic, oleic, elaidic acid chloride and the like.The thus monoacylated compounds are subjected to appropriatedeprotection treatment to remove the side chain 4-hydroxy protectinggroup which can be done in a highly selective manner with such reagents,depending on the regioselective protecting group, as HF/pyridine and thelike and manipulation of the reaction conditions, viz reagentconcentration, speed of addition, temperature and solvent etc, aselaborated above. The then free side chain 4-hydroxy group is acylatedwith the activated α-amino acid in a similar way as described in schemeA above.

Additional techniques for introducing the amino acid ester of R₁/R₂, forinstance in schemes A, B, C or D herein include the2-oxa-4-aza-cycloalkane-1,3-dione method described in internationalpatent application no. WO 94/29311.

Additional techniques for introducing the fatty acid ester of R₁/R₂, forinstance in schemes A, B, C or D herein include the enzymatic routedescribed in Preparative Biotransformations 1.11.8 (Ed S M Roberts, JWiley and Son, NY, 1995) with a lipase such as SP 435 immobilizedCandida antarcticus (Novo Nordisk), porcine pancreatic lipase or Candidarugosa lipase. Enzymatic acylation is especially convenient where it isdesired to avoid N-protection and deprotection steps on the other acylgroup or the purine 2-amine.

An alternative route to compounds of Formula I in which R₃ is hydrogenis to 6-activate the corresponding guanine compound of Formula I(wherein the amino acid ester moiety of R₁/R₂ is optionally protectedwith conventional N-protecting groups such as BOC) with an activatinggroup such as halo. The thus activated 6-purine is subsequently reducedto purine, for instance with a palladium catalyst and deprotected to thedesired 6-deoxy H2G di-ester.

A further aspect of the invention thus provides a method for thepreparation of the compounds of formula I comprising

a) optionally N-protecting the purine 2 and/or 6 positions of a compoundof formula I wherein R₁ and R₂ are each hydrogen;

b) regioselectively acylating the compound of Formula I at the sidechain 4-hydroxy group with either

-   -   i) an optionally N-protected valine or isoleucine group,    -   ii) an optionally substituted, saturated or monounsaturated        C₃-C₂₁COOH derivative, or    -   iii) a regioselective protecting group;        c) acylating at the side chain 2-hydroxymethyl group with    -   i) an optionally N-protected valine or isoleucine derivative, or    -   ii) an optionally substituted, saturated or monounsaturated        C₃-C₂₁COOH derivative;        d) replacing the regioselective protecting group at R₁, if        present, with    -   i) an optionally N-protected valine or isoleucine derivative; or    -   ii) an optionally substituted, saturated or monounsaturated        C₃-C₂₁COOH derivative; and        e) deprotecting the resulting compound as necessary.

Schemes A and B above employ selective acylation to stepwise add theamino acid and fatty acid esters. An alternative process for thepreparation of the compounds of formula I starts with a diacylated H2Gderivative, wherein both the acyl groups are the same, and employsselective removal of one of the acyl groups to obtain a monoacylintermediate which is then acylated with the second, differing, acylgroup in the same manner as Schemes A and B above.

Accordingly a further aspect of the invention provides a method for thepreparation of a compound of the formula I, as defined above, whichmethod comprises

A) the monodeacylation of a diacylated compound corresponding to formulaI wherein R₁ and R₂ are both a valyl or isoleucyl ester (which isoptionally N-protected) or are R₁ and R₂ are both —C(═O)C₃-C₂₁ saturatedor monounsaturated, optionally substituted alkyl; andB) acylating the thus liberated side chain 4-hydroxy or side chain2-hydroxymethyl group with the corresponding valyl, isoleucyl or—C(═O)C₃-C₂₁ saturated or monounsaturated, optionally substituted alkyl;andC) deprotecting as necessary.

This alternative process has the advantage that the preparation of thediacylated H2G derivative is facile and requires little or nopurification steps. Selective removal of one only of the acyl groups ofa diacylated H2G derivative can be achieved by manipulating the reactionconditions, in particular the temperature, rate of reactant addition andchoice of base.

Compounds amenable to this alternative synthesis route are thus of theformula:

where R₁ and R₂ are valyl or isoleucyl (which are optionallyN-protected) or a —C(═O)C₃-C₂₁ saturated or monounsaturated, optionallysubstituted alkyl; and R₃ is OH or H.

For ease of synthesis in this alternative route, it is preferred that R₁and R₂ are both initially identical and are most preferably the sameamino acid ester. Such a di-amino acid ester will generally beN-protected during its preparation and may be used directly in thiscondition in the selective deacylation step. Alternatively, such anN-protected di-aminoacylated H2G derivative may be deprotected andoptionally reprotected, as described below. The unprotected di-aminoacylH2G derivative thus comprises one of the following compounds:

-   (R)-9-[2-(L-isoleucyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,-   (R)-9-[2-(L-valyloxymethyl)-4-(L-valyloxy)butyl]guanine,-   (R)-2-amino-9-[4-(L-isoleucyloxy)-2-(L-isoleucyloxymethyl)butyl]purine,    and-   (R)-2-amino-9-[4-(L-valyloxy)-2-(L-valyloxymethyl)butyl]purine.

These unprotected H2G diacylated derivatives can be directly subject toselective deacylation of one of the acyl groups (typically the sidechain 4-position acyl) followed by enzymatic acylation of the liberated4-hydroxy as described above. Alternatively, the unprotected H2Gdiacylated derivative can be re-protected and then subjected to theselective deacylation, followed in turn by conventional acylation withthe fatty acid ester, as described in Schemes A and B. Conveniently,such a reprotection step is done with a different N-protecting group,having properties appropriate to the subsequent acylation. For example,it is convenient to employ a lipophilic N-protecting group, such as Fmocwhen preparing a di-amino acid H2G derivative, as the lipophilic natureof the protecting group assists with separation of the acylatedproducts. On the other hand, the lipophilic nature of Fmoc is of lessutility when conducting an acylation with a fatty acid, and thus it isconvenient to reprotect a diacylated H2G with an alternativeN-protecting group such as BOC.

It will also be apparent that the preparation of the compounds offormula I can commence with the novel monoacylated intermediates of stepb i), ii) or iii) in the above defined first method aspect of theinvention. These compounds are thus of the formula:

where one of R₁ and R₂ is

-   -   i) —C(O)CH(CH(CH₃)₂)NH₂ or —C(O)CH(CH(CH₃)CH₂CH₃)NH₂    -   ii) a —C(═O)C₃-C₂₁ saturated or monounsaturated, optionally        substituted alkyl, or    -   iii) a regioselective protecting group;    -   the other of R₁ and R₂ is hydrogen; and    -   R₃ is OH or H;

Useful compounds thus include:

-   (R)-9-[2-hydroxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,-   (R)-9-[2-hydroxymethyl-4-(trityloxy)butyl]guanine,-   (R)-9-[2-hydroxymethyl-4-(9-(9-phenyl)xanthenyloxy)butyl]guanine,-   (R)-9-[2-hydroxymethyl-4-(1,1-bis(4-methylphenyl)-1′-pyrenylmethyloxy)butyl]guanine,-   (R)-9-[2-hydroxymethyl-4-(decanoyloxy)butyl]guanine,-   (R)-9-[2-hydroxymethyl)-4-(dodecanoyloxy)butyl]guanine,-   (R)-9-[2-hydroxymethyl-4-(tetradecanoyloxy)butyl]guanine,-   (R)-9-[2-hydroxymethyl)-4-(hexadecanoyloxy)butyl]guanine,-   (R)-9-[2-hydroxymethyl-4-(octadecanoyloxy)butyl]guanine,-   (R)-9-[2-hydroxymethyl)-4-(eicosanoyloxy)butyl]guanine,-   (R)-9-[2-hydroxymethyl-4-(docosanoyloxy)butyl]guanine,-   (R)-9-[4-hydroxy-2-(decanoyloxymethyl)butyl]guanine,-   (R)-9-[4-hydroxy-2-(dodecanoyloxymethyl)butyl]guanine,-   (R)-9-[4-hydroxy-2-(tetradecanoyloxymethyl)butyl]guanine,-   (R)-9-[4-hydroxy-2-(hexadecanoyloxymethyl)butyl]guanine,-   (R)-9-[4-hydroxy-2-(octadecanoyloxymethyl)butyl]guanine,-   (R)-9-[4-hydroxy-2-(eicosanoyloxymethyl)butyl]guanine,-   (R)-9-[4-hydroxy-2-(docosanoyloxymethyl)butyl]guanine,-   (R)-9-[2-hydroxymethyl-4-(L-valyloxy)butyl]guanine,-   (R)-9-[2-hydroxymethyl)-4-(L-isoleucyloxy)butyl]guanine,-   (R)-9-[4-hydroxy-2-(L-isoleucyloxymethyl)butyl]guanine,-   (R)-9-[4-hydroxy-2-(L-valyloxymethyl)butyl]guanine.-   (R)-2-amino-9-[2-hydroxymethyl-4-(L-valyloxy)butyl]purine,-   (R)-2-amino-9-[2-hydroxymethyl)-4-(L-isoleucyloxy)butyl]purine,-   (R)-2-amino-9-[4-hydroxy-2-(L-isoleucyloxymethyl)butyl]purine, and-   (R)-2-amino-9-[4-hydroxy-2-(L-valyloxymethyl)butyl]purine.

Regioselectively protected, sidechain 4-hydroxy intermediates from stepc) of the above described first method aspect of the invention are alsonovel compounds. Useful compounds thus include:

-   (R)-9-[2-decanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,-   (R)-9-[2-dodecanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,-   (R)-9-[2-tetradecanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,-   (R)-9-[2-hexadecanoyloxymethyl-4-(t-butyldiphenylchlorosilane)butyl]guanine,-   (R)-9-[2-octadecanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,-   (R)-9-[2-eicosanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,-   (R)-9-[2-docosanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,

An alternative process for the preparation of compounds of the inventionof the formula I wherein R₃ is —OH is shown in Scheme C.

Referring to Scheme C, malonate 1 (R₄ and R₅ are lower alkyl or benzylor the like) is alkylated by reaction with from about 0.5 to about 2.0molar equivalents of acetal 2 (R₆ and R₇ are lower alkyl or benzyl andthe like or R₆ and R₇ taken together are —CH₂CH₂— or —CH₂CH₂CH₂— or—CH₂CH₂CH₂CH₂— and X₁ is a leaving group (for example, Cl, Br or I, or asulfonate such as methanesulfonate, triflate, p-toluenesulfonate,benzenesulfonate and the like)) in the presence of from about 0.5 toabout 2.0 molar equivalents of a base (for example, potassium t-butoxideor sodium ethoxide or NaH or KH and the like) in an inert solvent (forexample, DMF or THF or dioxane or dioxolane or N-methylpyrrolidone andthe like) at a temperature of from about −40° C. to about 190° C. toprovide alkylated malonate 3.

Reduction of 3 with from about 0.5 to about 4.0 molar equivalents of anester to alcohol reducing agent (for example, LiBH₄ or Ca(BH₄)₂ or NaBH₄or LiAlH₄ and the like) in an inert solvent (for example, THF or methylt-butyl ether or t-BuOH and the like) at a temperature of from about−20° C. to about 100° C. provides diol 4. Enzymatic esterification of 4by reaction with from about 1.0 to about 20.0 molar equivalents of avinyl ester 5 (R₈ is C₃-C₂₁ saturated or monounsaturated, optionallysubstituted alkyl) in the presence of a lipase (for example, LipasePS-30 or Lipase PPL or Lipase CCL and the like) or a phospholipase (forexample phospholipase D and the like) provides the desired stereoisomerof ester 6. This reaction can be carried out in the absence of solventor in the presence of an inert solvent (for example, methyl t-butylether or toluene or hexane and the like). The reaction is carried out ata temperature of from about −20° C. to about 80° C.

The alcohol substituent of 6 is converted to a leaving group (forexample, a halogen or a sulfonate) by reaction with a halogenating agent(for example NBS/P(Ph)₃ or NCS/P(Ph)₃ or POCl₃ or NCS/P(Ph)₃/Nal inacetone and like) in an inert solvent (for example, methylene chlorideor toluene or ethylacetate and the like) or by reaction with from about0.8 molar equivalents to about 2.0 molar equivalents of a sulfonylhalide (for example, benzenesulfonylchloride, toluenesulfonylchloride ormethane sulfonylchloride and the like) in the presence of from about 1.0to about 4.0 molar equivalents of a base (for example, triethylamine orpotassium carbonate or pyridine or dimethylaminopyridine orethyldiisopropylamine and the like) in an inert solvent (for examplemethylene chloride or toluene or ethylacetate or pyridine or methylt-butyl ether and the like) at a temperature of from about −25° C. toabout 100° C. to provide ester 7. (X₂ is a halogen or sulfonate leavinggroup).

Reaction of 7 with from about 0.9 to about 2.0 molar equivalents of2-amino-4-chloropurine 8 in the presence of from about 1.0 to about 6.0molar equivalents of a base (for example, potassium carbonate or NaH orKH or NaOH or KOH or lithium diisopropylamide and the like) in an inertsolvent (for example, DMF or THF or acetonitrile or N-methylpyrrolidoneor ethanol and the like) at a temperature of from about −25° C. to about140° C. provides substituted purine 9.

Alternatively Mitsunobu coupling (for example P(Ph)₃/diethylazidocarboxylate) of alcohol 6 with 2-amino-4-chloropurine 8 provides 9.

Reaction of 9 with from about 2.0 to about 20 molar equivalents of analcohol R₉OH (R₉ is an alcohol protecting group such as benzyl and thelike) in the presence of from about 1.0 to about 6.0 molar equivalentsof a base (for example, potassium t-butoxide or potassium carbonate orNaH or KH or lithium diisopropylamide and the like) in an inert solvent(for example, THF or DMF and the like) at a temperature of from about−25° C. to about 150° C. provides alcohol 10.

Removal of the alcohol protecting group R₉ of 10 (for example, bycatalytic hydrogenation in an inert solvent such as ethanol or benzylalcohol or methanol or THF and the like in the presence of anhydrogenation catalyst such as Pd/C or Pd(OH)₂ and the like) providessubstituted guanine 11.

Esterification of 11 by reaction with a) from about 0.8 to about 2.0molar equivalents of R₁₀COOH and a coupling agent (for example DCC/DMAP)and the like in an inert solvent (for example THF or DMF and the like)or b) from about 0.8 to about 2.0 molar equivalents of an activatedderivative of R₁₀COOH (for example, the acid chloride orN-hydroxysuccinimide ester or R₁₀C(O)OC(O)R₁₀ and the like) in thepresence of from about 0 to about 3.0 molar equivalents of a base (forexample, pyridine or triethylamine or ethyldiisopropylamine or DBU orpotassium carbonate and the like) in an inert solvent (for example,methylene chloride or THF or pyridine or acetonitrile or DMF and thelike) at a temperature of from about −25° C. to about 100° C. providesester 12.

The acetal substituent of 12 is deprotected and the resulting aldehydeis reduced by first reacting 12 with from about 0.1 to about 10.0 molarequivalents of an acid (for example, triflic acid or HCl or acetic acidor sulfuric acid and the like) in an inert solvent (for example, THF/H₂Oor methylene chloride/H₂O or ethylacetate/H₂O or ethanol/H₂O ormethanol/H₂O and the like) at a temperature of from about −25° C. toabout 100° C. To the crude reaction mixture is added from about 0.1 toabout 10.0 molar equivalents of a base (for example, sodium bicarbonateor potassium carbonate or triethylamine or pyridine or KOH and thelike), additional inert solvent (for example, THF and or methylenechloride or ethylacetate or methyl t-butyl ether or isopropanol and thelike) and from about 0.3 to about 5.0 molar equivalents of an aldehydereducing agent (for example, sodium borohydride or RaNi/H₂ and the like)at a temperature of from about −25° C. to about 100° C. to providealcohol 13.

Reaction of 13 with from about 0.8 to about 3.0 molar equivalents ofN-protected amino acid P₁NHCH(R₁₁)COOH or an activated derivativethereof (P₁ is an N-protecting group and R₁₁ is isopropyl or isobutyl)in an inert solvent (for example, THF or dioxane or dioxolane or DMF ormethylene chloride and the like) at a temperature of from about 25° C.to about 100° C. provides alcohol 14. N-deprotection of 14 provides thecompound of the invention of formula I wherein R₃ is —OH.

Alternatively compound 13 can be reacted with the symmetrical anhydridederived from P₁NHCH(R₁₁)COOH (i.e. P₁NHCH(R₁₁)C(O)O—C(O)CH(R₁₁)NHP₁) toprovide 1 wherein R₃ is OH.

Another alternative process for the preparation of compounds wherein R₃is —OH is shown in Scheme D.

Malonate 1 (R₄ and R₅ are lower alkyl or benzyl and the like) isalkylated with from about 0.5 to about 2.0 molar equivalents of ether 15wherein X₁ is a leaving group (for example Cl, Br or I, or a sulfonatesuch as methane sulfonate, triflate, p-toluenesulfonate,benzenesulfonate and the like) and R₁₂ is —CH(Ph)₂, —C(Ph)₃ or—Si(t-Bu)(Me)₂ and the like (Ph=phenyl) in the presence of from about0.5 to about 2.0 molar equivalents of a base (for example potassiumt-butoxide or sodium ethoxide or NaH or KH and the like) in an inertsolvent (for example DMF or THF or dioxane or dioxolane or N-methylpyrrolidinone and the like) at a temperature of from about −40° C. toabout 190° C. to provide alkylated malonate 16.

Reduction of 16 with from about 0.5 to about 4.0 molar equivalents of anester to alcohol reducing agent (for example LiBH₄ or Ca(BH₄)₂ or NaBH₄or LiAlH₄ and the like) in an inert solvent (for example THF or methylt-butyl ether or ethanol or t-butanol and the like) at a temperature offrom about −20° C. to about 100° C. provides diol 17. Enzymaticesterification of 17 by reaction with from about 1.0 to about 20.0 molarequivalents of a vinyl ester 5 (R₈ is C₃-C₂₁ saturated ormonounsaturated, optionally substituted alkyl) in the presence of alipase (for example, Lipase PS-30 or Lipase PPL or Lipase CCL and thelike) or a phospholipase (for example phospholipase D and the like)provides the desired stereoisomer of ester 18. The reaction can becarried out in the absence of solvent or in the presence of an inertsolvent (for example methyl t-butyl ether or toluene or hexane or thelike). The reaction is carried out at a temperature of from about −20°C. to about 80° C.

The alcohol substituent of 18 is converted to a leaving group (forexample a halogen or sulfonate) by reaction with a halogenating agent(for example NBS/P(Ph)₃ or NCS/P(Ph)₃ or POCl₃ or NCS/P(Ph)₃/Nal inacetone and the like) in an inert solvent (for example methylenechloride or toluene or ethylacetate and the like) or by reaction withfrom about 0.8 molar equivalents to about 2.0 molar equivalents of asulfonyl halide (for example benzenesulfonylchloride,toluenesulfonylchloride or methane sulfonylchloride and the like) in thepresence of from about 1.0 to about 4.0 molar equivalents of a base (forexample triethylamine or potassium carbonate or pyridine or methylt-butyl ether and the like) at a temperature of from about −25° C. toabout 100° C. to provide ester 19. (X₂ is a halogen or sulfonate leavinggroup).

Reaction of 19 with from about 0.9 to about 2.0 molar equivalents of2-amino-4-chloropurine 8 in the presence of from about 1.0 to about 6.0molar equivalents of a base (for example potassium carbonate or NaH orKH or NaOH or KOH or lithium diisopropylamide and the like) in an inertsolvent (for example DMF or THF or acetonitrile or N-methylpyrrolidoneor ethanol and the like) at a temperature of from about −25° C. to about140° C. provides substituted purine 20.

Alternatively, Mitsunobu coupling (for example, P(PH)₃/diethylazidocarboxylate) of alcohol 18 with 2-amino-4-chloropurine 8 provides20.

Reaction of 20 with from about 2.0 to about 20.0 molar equivalents of analcohol R₉OH (R₉ is an alcohol protecting group such as benzyl and thelike) in the presence of from about 1.0 to about 6.0 molar equivalentsof a base (for example, potassium t-butoxide or potassium carbonate orNaH or KH or lithium diisopropylamide and the like in an inert solvent(for example, THF or DMF and the like) at a temperature of from about−25° C. to about 150° C. provides alcohol 21.

Removal of the alcohol protecting group R₉ of 21 (for example bycatalytic hydrogenation in an inert solvent such as ethanol or benzylalcohol or methanol or THF and the like in the presence of anhydrogenation catalyst such as Pd/C or Pd(OH)₂ and the like) providessubstituted guanine 22.

The ether substitutent of 23 is deprotected by reaction with a) areducing agent (for example, HCO₂H and Pd/C and the like) wherein R₁₂ is—CH(Ph)₂ or —C(Ph)₃, or b) a desilylating agent (for example Bu₄NF andthe like) wherein R₁₂ is —Si(t-Bu)(Me)₂ and the like to provide 13.

Alcohol 13 can be converted to 1 as outlined in scheme C.

An additional alternative involves enzymatic esterification of alcohol 4or 17 with the vinyl ester CH₂═CH—OC(O)R₁₀ (i.e. R₈═R₁₀ in Schemes C andD) to directly incorporate into 6 or 18 the desired carboxylic acidester of the final product I. This allows the elimination of the esterhydrolysis and reesterification involved in going from 9 to 12 or from20 to 23.

The processes of Schemes C and D are characterized by the fact that eachof the hydroxyl groups of the acyclic side chain is differentiated bythe use of different hydroxy protecting groups or precursor groups. Thisallows the selective acylation of each of the hydroxy groups with eitheran amino acid or a fatty acid group.

Schemes C and D have been illustrated and described with reference toembodiments of the invention wherein R₁ is derived from an amino acidand R₂ is derived from a fatty acid. However, it will be apparent thatrespective converse schemes will apply to compounds where R₁ is derivedfrom a fatty acid and R₂ is derived from an amino acid.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be illustrated by way of example only withreference to the following non-limiting Examples, comparative examplesand the accompanying Figures, in which:

FIG. 1 depicts plasma H2G levels as a function of time in cynomolgusmonkeys administered with a compound of the invention or with analternative prodrug derivative of H2G, as further explained inBiological Example 3; and

FIG. 2 depicts survival as a function of time for Herpes simplexinfected mice administered with various doses of a compound of theinvention or a prior art antiviral, as further explained in BiologicalExample 4.

EXAMPLE 1 (R)-9-[2-(Stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine

This example illustrates the application of preparation scheme A.

a) (R)-9-[4-(N-tert-Butoxycarbonyl-L-valyloxy)-2-(hydroxymethyl)butyl]guanine

H2G (5 g, 19.7 mmol) was dissolved in DMF (300 ml) under heating and wascooled to room temperature before addition of N-t-Boc-L-valine (5.58 g,25.7 mmol), DMAP (0.314 g, 2.57 mmol) and DCC (6.52 g, 31.6 mmol). Themixture was stirred at room temperature for 24 h and was then filtered.The product was chromatographed on silica gel and eluted withCH₂Cl₂/MeOH to give 2.4 g of the desired intermediate product.

¹H-NMR (250 MHz, DMSO-d₆): δ 0.95 (d, 6H), 1.47 (s, 9H), 1.5-1.8 (m,2H), 1.96-2.20 (m, 2H), 3.40 (m, 2H), 3.91 (t, 1H), 4.05 (m, 2H), 4.21(t, 2H), 4.89 (t, 1H), 6.6 (br s, 2H), 7.27 (d, 1H), 7.75 (s, 1H), 10.7(br s, 1H).

b) (R)-9-[4-(N-tert-Butoxycarbonyl-L-valyloxy)-2-(stearoyloxymethyl)butyl]guanine

The product from step a) (185 mg, 0.41 mmol) was dissolved in pyridine(5 ml), the solution was cooled in an ice bath and stearoyl chloride(179 μl, 0.531 mmol) was added. The solution was kept in the ice bathfor 2 h, then at room temperature for 1 h. It was then evaporated andchromatographed on silica gel. It was eluted withdichloromethane/methanol to give 143 mg of the desired intermediateproduct.

c) (R)-9-[2-(Stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine

The product from step b) (138 mg, 0.192 mmol) was cooled in an ice bathand trifluoroacetic acid (5 ml) was added. The solution was kept in theice bath for 45 minutes and was then evaporated to give an oil. Water(0.5 to 1 ml) was added and evaporated twice. The residue was once moredissolved in water (5 ml), filtered and freeze-dried to give 148 mg ofthe desired product as the bistrifluoracetate salt.

¹H NMR (250 MHz, DMSO-d₆): 0.97 (t, 3H), 1.05 (dd, 6H), 1.34 (br s,28H), 1.59 (m, 2H), 1.80 (m, 2H), 2.25 (m, 1H), 2.36 (t, 2H), 2.50 (m,1H), 3.98-4.18 (m, 5H), 4.35 (t, 2H), 6.6 (br s, 2H), 8.0 (br s, 1H),8.4 (br s, 3H), 10.9 (br s, 1H).

EXAMPLE 2 (R)-9-[2-(Myristoyloxymethyl)-4-(L-valyloxy)butyl]guanine

The titled compound was obtained as the bistrifluoracetate salt in amanner analogous to Example 1 using myristoyl chloride instead ofstearoyl chloride in step b).

¹H NMR (250 MHz, DMSO-d₆): δ 0.97 (t, 3H), 1.05 (dd, 6H), 1.34 (br s,20H), 1.57 (m, 2H), 1.78 (m, 2H), 2.24 (m, 1H), 2.35 (t, 2H), 2.51 (m,1H), 3.97-4.20 (m, 5H), 4.36 (t, 2H), 6.8 (br s, 2H), 8.2 (br s, 1H),8.5 (br s, 3H), 11.1 (br s, 1H).

EXAMPLE 3 (R)-9-[2-(Oleoyloxymethyl)-4-(L-valyloxy)butyl]guanine

The titled compound was obtained as the bistrifluoroacetyl salt in amanner analogous to Example 1 using oleoyl chloride instead of stearoylchloride in step b).

¹H NMR (250 MHz, DMSO-d₆): 0.96 (t, 3H), 1.05 (dd, 6H), 1.35 (br s,20H), 1.59 (m, 2H), 1.76 (m, 2H), 2.09 (m, 4H), 2.24 (m, 1H), 2.35 (t,2H), 2.50 (m, 1H), 3.97-4.17 (m, 5H), 4.35 (t, 2H), 5.43 (t, 2H), 6.7(br s, 2H), 8.0 (br s, 1H), 8.5 (br s, 3H), 11.1 (br s, 1H).

EXAMPLE 4 (R)-9-[2-(Butyryloxymethyl)-4-(L-valyloxy)butyl]guanine a)(R)-9-[4-(N-tert-Butoxycarbonyl-L-valyloxy)-2-(butyryloxymethyl)butyl]guanine

DCC (110 mg, 0.53 mmol) was dissolved in dichloromethane (10 ml) andbutyric acid (82 mg, 0.93 mmol) was added. After 4 hours at roomtemperature the mixture was filtered and the filtrate was evaporated.The residue was dissolved in pyridine (5 ml) and(R)-9-[4-(N-tert-Butoxycarbonyl-L-valyloxy)-2-hydroxymethylbutyl]guanine(200 mg, 0.44 mmol) (Example 1, step a) was added. The mixture wasstirred for 120 hours at room temperature. According to TLC the reactionwas incomplete and more anhydride was made using the procedure above.This anhydride was added and the mixture was stirred for an additional20 hours. The reaction mixture was evaporated and chromatographed firston silica gel and then on aluminium oxide, in both cases eluted withdichloromethane/methanol to give 79 mg of the intermediate product.

b) (R)-9-[2-(Butyryloxymethyl)-4-(L-valyloxy)butyl]guanine

The intermediate product of step a was deprotected in a manner analogousto Example 1, step 3 to give 84 mg of the desired product as thebistrifluoracetate salt.

¹H NMR (250 MHz, D₂O): δ 0.88 (t, 3H), 1.06 (dd, 6H), 1.53 (m, 2H), 1.93(q, 2H), 2.25 (t, 2H), 2.36 (m, 1H), 2.60 (m, 1H), 4.06 (d, 1H),4.14-4.30 (m, 2H), 4.43 (m, 4H), 8.99 (br s, 1H).

EXAMPLE 5 (R)-9-[2-(Decanoyloxymethyl)-4-(L-valyloxy)butyl]guanine

The titled compound was obtained as the bistrifluoroacetate salt in amanner analogous to Example 1 using decanoyl chloride instead ofstearoyl chloride in step b.

¹H NMR (250 MHz, D₂O): δ 0.90 (m, 3H), 1.01 (d, 6H), 1.28 (br s, 12H),1.5 (m, 2H), 1.8 (m, 2H), 2.3 (m, 3H), 2.5 (m, 1H), 4.0-4.4 (m, 7H), 8.1(br s, 1H).

EXAMPLE 6 (R)-9-[2-Docosanoyloxymethyl-4-(L-valyloxy)butyl]guanine

The titled compound was obtained as the bistrifluoroacetate salt in amanner analogous to Example 1 but using in step b the DMAP/DCCconditions of Example 1, step a) in conjunction with docosanoic acid inplace of the N-t-Boc-L-valine and a mixture of DMF and dichloromethaneas solvent.

¹H NMR (250 MHz, DMSO-d₆): δ 0.97 (t, 3H), 1.05 (dd, 6H), 1.34 (br s,36H), 1.58 (m, 2H), 1.77 (m, 2H), 2.24 (m, 1H), 2.35 (t, 2H), 2.50 (m,1H), 3.97-4.17 (m, 5H), 4.35 (t, 2H), 6.7 (br s, 2H), 8.1 (br s, 1H),8.4 (br s, 3H), 11.0 (br s, 1H).

EXAMPLE 7 R-9-[4-(L-Isoleucyloxy)-2-(stearoyloxymethyl)butyl]guanine

This example illustrates the application of preparative scheme B.

a) (R)-9-[2-hydroxymethyl 4-(t-butyldiphenylsilyloxy) butyl]guanine

H2G (2 g, 8 mmole) was coevaporated with dry DMF two times and was thensuspended in dry DMF (120 ml) and pyridine (1 ml). To the suspension wasadded dropwise t-butyldiphenylchlorosilane (2.1 ml, 8.2 mmole) indichloromethane (20 ml) at 0° C. over a period of 30 min. The reactionmixture became a clear solution at the completion of the dropwiseaddition. The reaction continued at 0° C. for two hours and was thenkept at 4° C. overnight. Methanol (5 ml) was added to the reaction.After 20 min at room temperature, the reaction mixture was evaporated toa small volume, poured into aqueous sodium hydrogen carbonate solutionand extracted with dichloromethane two times. The organic phase wasdried over sodium sulphate and evaporated in vacuo. The product wasisolated by silica gel column chromatography using amethanol/dichloromethane system with a stepwise increasing MeOHconcentration. The product was eluted with 7% MeOH in CH₂Cl₂ to yield1.89 g.

b) (R)-9-[2-(Stearoyloxymethyl)-4-(t-butyldiphenylsilyloxy)butyl]guanine

(R)-9-[2-Hydroxymethyl 4-(t-butyldiphenylsilyloxy)butyl]guanine (2.31 g,5 mmole) was coevaporated with dry pyridine twice and dissolved inpyridine (20 ml). To the solution was slowly added dropwise stearoylchloride (1.86 ml, 5.5 mmole, technical grade) in dichloromethane (2 ml)at −5° C. The reaction was kept at the same temperature for 1 hr andthen at 5° C. for 2 hr. The reaction was monitored by TLC. Additionalstearoyl chloride (0.29 ml) at −5° C. was added due to incompletion ofreaction. After 30 min at 5° C., methanol (3 ml) was added and thereaction mixture stirred for 20 min. It was then poured into aqueoussodium hydrogen carbonate solution, and extracted with dichloromethane.The organic phase was dried and the product purified by silica gelcolumn chromatography with stepwise increasing MeOH, eluting with 3.5%MeOH in CH₂Cl₂. (Yield 2.7 g).

c) (R)-9-[(4-Hydroxy-2-(stearoyloxymethyl)butyl]guanine

(R)-9-[2-(Stearoyloxymethyl)-4-(t-butyldiphenylsilyloxy)butyl]guanine(2.7 g, 3.56 mmole) was dissolved in dry THF (30 ml) and hydrogenfluoride-pyridine (1.5 ml) added to the solution. The reaction was keptat 4° C. overnight and monitored by TLC. The reaction reached about 80%conversion. Additional HF-pyridine was added (0.75 ml). After 4 hr, TLCshowed that the starting material had disappeared. The reaction mixturewas concentrated in vacuo without raising the temperature and morepyridine (5 ml) was added and evaporated again. The product was isolatedby silica gel column chromatography. (Yield 1.26 g).

d) (R)-9-[4-(N—BOC-L-isoleucyloxy)-2-(stearoyloxymethyl)butyl]guanine

(R)-9-[4-Hydroxy-2-(stearoyloxymethyl)butyl]guanine (135 mg, 0.26 mmole)and N—BOC-L-isoleucine (180 mg, 0.78 mmole) were coevaporated with dryDMF twice and dissolved in the same solvent (3.5 ml). To the solutionwas added 1,3-dicyclohexylcarbodiimide (160 mg, 0.78 mmole) and4-dimethylaminopyridine (4.8 mg, 0.039 mmole). After reaction for 18hours, the reaction mixture was filtered through Celite and worked up ina conventional manner. The product was isolated by silica gel columnchromatography, eluting at 5% MeOH in CH₂Cl₂. (Yield 160 mg)

e) (R)-9-[4-(L-Isoleucyloxy)-2-(stearoyloxymethyl)-butyl]guanine

(R)-9-[4-(N—BOC-L-isoleucyloxy)-2-(stearoyloxymethyl)butyl]guanine (150mg, 0.205 mmole) from step d) was treated with trifluoroacetic acid (3ml) at 0° C. for 20 min. The solution was evaporated in vacuo. Theresidue was coevaporated with toluene twice and kept under vacuum forseveral hours. The residue was dissolved in MeOH (2 ml) and evaporatedto give the trifluoracetate salt as a glass-like product (Yield 191 mg).

H¹NMR (DMSO-d6+D₂O): δ 8.35 (s, 1H, base), 4.21 (t, 2H, H-4), 4.10 (d,2H) 3.96 (d, 2H), 3.90 (d, 1H, isoleucine), 2.48 (m, 1H, H-2), 2.15 (2H,stearoyl), 1.85 (m, 1H, isoleucine), 1.68 (m, 2H), 1.48 (m, 4H), 1.68(m, 28H), 0.81 (m, 9H).

EXAMPLE 8 (R)-9-[2-(Decanoyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine

The title compound was obtained as the bistrifluoroacetyl salt in amanner analogous to Example 7 using decanoyl chloride instead ofstearoyl chloride in step b).

¹H NMR (DMSO-d6): δ 11.1 (s, 11H, NH), 8.35 (s, br, 3H), 8.28 (s, 11H,base), 6.75 (s, 2H, NH₂), 4.23 (t, 2H), 4.07 (d, 2H), 4.05 (m, 3H), 2.4(m, 1H), 2.21 (t, 2H), 1.83 (m, 1H), 1.66 (m, 2H), 1.45 (m, 2H), 1.39(m, 2H), 1.22 (s, 12H), 0.84 (m, 9H).

EXAMPLE 9 (R)-9-[4-(L-Isoleucyloxy)-2-(myristoyloxymethyl)butyl]guanine

The title compound was obtained as the bistrifluoroacetyl salt in amanner analogous to Example 1 using N—BOC-L-isoleucine instead ofN—BOC-valine in step a) and myristoyl chloride in step b).

¹H-NMR (DMSO-d6): δ 10.99 (s, 1H), 8.34 (br s, 3H) 8.15 (s, 11H), 6.67(br s, 2H), 4.23 (t, 2H), 4.05 (d, 2H), 3.97 (m, 3H), 2.48 (m, 1H), 2.20(t, 2H), 1.85 (m, 1H), 1.65 (m, 2H), 1.41 (m, 4H), 1.23 (s, 20H), 0.85(m, 9H).

EXAMPLE 10(R)-9-[2-(4-Acetylbutyryloxymethyl-4-(L-valyloxy)butyl]guanine

The titled compound was obtained as the bistrifluoroacetate salt in amanner analogous to Example 1 but using in step b) the DCC/DMAPconditions of Example 1, step a) in conjunction with 4-acetylbutyricacid instead of N-t-Boc-L-valine.

¹H-NMR (250 MHz, DMSO-d₆): δ 1.05 (dd, 6H), 1.77 (m, 4H), 2.19 (s, 3H),2.24 (m, 1H), 2.36 (t, 2H), 2.44-2.60 (m, 3H), 3.95-4.20 (m, 5H), 4.36(m, 2H), 6.8 (br s, 2H), 8.3 (br s, 1H), 8.5 (br s, 3H), 11.1 (br s,1H).

EXAMPLE 11 (R)-9-[2-Dodecanoyloxymethyl-4-(L-valyloxy)butyl]guanine

The titled compound was obtained as the bistrifluoroacetate salt in amanner analogous to Example 1 using dodecanoyl chloride instead ofstearoyl chloride in step b).

EXAMPLE 12 (R)-9-[2-Palmitoyloxymethyl-4-(L-valyloxy)butyl]guanine

The titled compound was obtained as the bistrifluoroacetate salt in amanner analogous to Example 1 using palmitoyl chloride instead ofstearoyl chloride in step b).

¹H-NMR (250 MHz, DMSO-d₆): δ 0.97 (t, 3H), 1.05 (m, 6H), 1.35 (br s,24H), 1.58 (m, 2H), 1.78 (m, 2H), 2.25 (m, 1H), 2.35 (t, 2H), 2.51 (m,1H), 3.97-4.18 (m, 5H), 4.35 (t, 2H), 6.7 (br s, 2H), 8.1 (br s, 1H),8.5 (br s, 3H), 11.0 (br s, 1H).

EXAMPLE 13 (R)-2-Amino-9-(2-stearoyloxymethyl-4-(L-valyloxy)butyl)purine

This example shows the deoxygenation of group R₁.

a)(R)-2-Amino-9-(2-stearoyloxymethyl-4-(N-tert-butoxycarbonyl-L-valyloxy)butyl)-6-chloropurine

To a solution of(R)-9-(2-stearoyloxymethyl-4-(N-tert-butoxycarbonyl-L-valyloxy)butyl)guaninefrom step 2 of Example 1 (646 mg, 0.9 mmole) in acetonitrile were addedtetramethylammonium chloride (427 mg, 2.7 mmole), N,N-diethylaniline(0.716 ml, 4.5 mmole) and phosphorous oxychloride (0.417 ml, 4.5 mmole).The reaction was kept under reflux and the progression monitored by TLC.After 3 hours the reaction mixture was evaporated in vacuo and theresidue was dissolved in dichloromethane, then poured into cold sodiumhydrogen carbonate aqueous solution. The organic phase was evaporatedand purified by silica gel column chromatography. Yield: 251 mg.

H¹-NMR (CDCL₃): δ 7.76 (1H, H-8), 5.43 (br, 2H, NH₂), 4.45-4.00 (m, 7H),2.53 (m, 1H), 2.28 (t 2H), 2.12 (m, 1H), 1.75 (m, 2H), 1.59 (m, 2H),1.43 (9H), 1.25 (m, 28H), 0.96 (d, 3H), 0.87 (m, 6H).

b)(R)-2-Amino-9-(2-stearoyloxmethyl-4-(N-tert-butoxycarbonyl-L-valyloxy)butyl)purine

To the solution of(R)-2-amino-9-(2-stearoyloxymethyl-4-(N-tert-butoxycarbonyl-L-valyloxy)butyl)-6-chloropurine(240 mg, 0.33 mmole) in methanol/ethyl acetate (6 ml, 3:1 V/V) wereadded ammonium formate (105 mg, 1.65 mmole) and 10% palladium on carbon(15 mg). The reaction was kept under reflux for 1 hour and rechargedwith ammonium formate (70 mg). After one hour more the TLC showedcompletion of the reaction and the mixture was filtered through Celiteand washed extensively with ethanol. The filtrate was evaporated andpurified by silica gel column. Yield: 193 mg.

H¹-NMR (CDCL₃): δ8.69 (s, 1H, H-6), 7.74 (s, 1H, H-8), 5.18 (br, s, 2H,NH₂), 4.45-4.01 (m, 7H), 2.55 (m, 1H), 2.28 (t, 2H), 2.10 (m, 11H), 1.75(m, 2H), 1.60 (m, 2H), 1.43 (s, 9H), 1.25 (s, 28H), 0.96 (d, 3H), 0.87(m, 6H).

c) (R)-2-Amino-9-(2-stearoyloxymethyl-4-(L-valyloxy)butyl)purine

(R)-2-Amino-9-(2-Stearoyloxmethyl-4-(N-tert-butoxycarbonyl-L-valyloxy)butyl)purine(180 mg, 0.26 mmole) was treated with trifluoroacetic acid (5 ml) at 0°C. for 40 min. It was then evaporated in vacuo and coevaporatedsuccessively with toluene and methanol. The residue was freeze-driedovernight to give 195 mg of the desired product.

¹H-NMR (DMSO-d6): δ 8.78 (s, 11H, H-6), 8.32 (br, 3H), 8.29 (s, 1H,H-8), 4.27 (t, 2H), 4.13 (d, 2H), 3.98 (t, 2H, 2H), 3.89 (m, 1H), 2.47(m, 1H), 2.18 (m, 3H), 1.43 (m, 2H), 1.23 (28H), 0.93 (m, 6H), 0.85 (t,3H).

EXAMPLE 14 Alternative preparation of(R)-9-[4-Hydroxy-2-(stearoyloxymethyl)butyl]guanine

a) Preparation of ethyl 4,4-diethoxy-2-ethoxycarbonyl-butyrate

Potassium tert-butoxide (141.8 g, 1.11 equiv.) was dissolved in dry DMF(1 L). Diethyl malonate (266 mL, 1.54 equiv.) was added over 5 minutes.Bromoacetaldehyde diethylacetal (172 mL, 1.14 mole) was added over 5minutes. The mixture was heated to 120° C. (internal temperature), andstirred at 120° C. for 5 hours. The mixture was allowed to cool to roomtemperature, poured into water (5 L), and extracted with methyltert-butyl ether (MTBE, 3×600 mL). The organic solution was dried overMgSO₄, filtered, concentrated, and distilled (0.5 mm, 95-140° C.) toyield the desired diester (244 g, 78%) as a colorless oil.

¹H NMR (CDCl₃) δ 1.19 (t, 6H), 1.28 (t, 6H), 2.22 (dd, 2H), 3.49 (m,2H), 3.51 (t, 1H), 3.65 (m, 2H) 4.20 (qd, 4H), 4.54 (t, 1H).

b) Preparation of 4,4-diethoxy-2-(hydroxymethyl)-butanol

LiBH4 (purchased solution, 2M in THF, 22.5 mL) and the product ofExample 14 step a) (5 g in 15 mL of THF, 18.1 mmol) were combined andwarmed to 60° C. and stirred at 60° C. for 4 hours. The reaction mixturewas allowed to cool to room temperature and the reaction vessel wasplaced in a cool water bath. Then triethanolamine (5.97 mL, 1 equiv.)was added at such a rate that the temperature of the reaction mixturewas maintained between 20-25° C. Brine (17.5 mL) was added at a ratesuch that gas evolution was controlled and the mixture was stirred for45 minutes at room temperature. The layers were separated, the organiclayer was washed with brine (2×15 mL). The combined brine washes wereextracted with MTBE (methyl tert-butyl ether, 3×20 mL). The combinedorganic extracts were evaporated and the residue was dissolved in MTBE(50 mL) and washed with brine (25 mL). The brine layer wasback-extracted with MTBE (3×25 mL). The combined organic extracts weredried over Na₂SO₄, filtered, and concentrated to yield the desired diol(3.36 g, 15.5 mmol, 97%) as a colorless oil.

¹H NMR (CDCl₃) δ 1.22 (t, 6H), 1.73 (dd, 2H), 1.92 (m, 1H), 2.67 (bs,2H), 3.52 (m, 2H), 3.69 (m, 2H), 3.72 (m, 4H), 4.62 (t, 1H).

c) Preparation of (2R)-2-acetoxymethyl-4,4-diethoxy-butanol

Into a 10 ml 1 neck round bottom flask was charged the product ofExample 14 step b) (3.84 g, 20 mmol), followed by addition of vinylacetate (2.6 g, 30 mmol) and finally Lipase PS 30 (69 mg, purchased from(Amano, Lombard, Ill.). The mixture was allowed to stir at ambienttemperature for 16 hours. Progress of the reaction was closely monitoredby TLC (2/1 hexane-EtOAc; stained with Ce₂(SO₄)₃ and charred on hotplate; r.f. of diol is 0.1, monoacetate is 0.3, bis acetate is 0.75).The reaction mixture was diluted with CH₂Cl₂ and filtered through a 5micron filter. The filter was washed with additional CH₂Cl₂. Thefiltrate was then concentrated in vacuo to afford the desired product.

d) Preparation of (2S)-2-acetoxymethyl-4,4-diethoxybutyltoluenesulfonate

Into a 100 mL 1-neck round bottom flask, equipped with a magnetic stirbar and septum under N2 was charged the crude product of Example 14 stepc) (4.62 g, 19 mmol), dry CH₂Cl₂ (20 mL) and Et₃N (5.62 mL, 40 mmol). Tothis solution was added tosyl chloride (4.76 g, 25 mmol). The resultingmixture was stirred at ambient temperature for 4 hours. Charged H₂O(0.27 g, 15 mmol) and stirred vigorously for 4 hours. The reactionmixture was diluted with 80 mL EtOAc and 50 mL H₂O and the aqueous layerwas separated. To the organic layer was added 75 ml of a 5% aq. solutionof KH₂PO₄. After mixing and separation of the layers, the aqueous layerwas removed. The organic layer was washed with 50 mL of saturated NaHCO₃solution, dried over Na₂SO₄, filtered and concentrated in vacuo to aconstant weight of 7.40 g of the desired product.

¹H NMR (CDCl₃) δ 1.17 (t, 6H); 1.62 (m, 2H); 1.94 (s, 3H); 2.19 (m, 1H);2.45 (s, 3H); 3.42 (m, 2H); 3.6 (m, 2H); 4.03 (m, 4H); 4.51 (t, 1H);7.36 (d, 2H); 7.79 (d, 2H).

e) Preparation of

Into a 50 mL 1 neck round bottom flask was charged the product ofExample 14 step d) (3.88 g, 10 mmol), anhydrous DMF (20 mL),2-amino-4-chloro-purine (2.125 g, 12.5 mmol) and K₂CO₃ (4.83 g). Theresulting suspension was stirred at 40° C. under a N₂ blanket for 20hours. The mixture was concentrated to remove most of the DMF on arotary evaporator. The residue was diluted with EtOAc (50 mL) and H₂O(50 mL). The reaction mixture was transferred to a separatory funnel,shaken and the aqueous layer was separated. The aqueous layer wasextracted with EtOAc (25 mL). The organic layers were combined andwashed with 5% KH₂PO₄ (75 mL). The organic layer was separated andwashed with H₂O (75 mL), brine (75 mL), dried over Na₂SO₄, filtered andconcentrated in vacuo to afford 3.95 g of crude product. The crudeproduct was slurried with 40 mL of methyl-t-butyl ether. This mixturewas stirred overnight at 4° C. and the mixture was filtered. Thefiltrate was concentrated to afford 3.35 g of the product as an oil(containing 2.6 g of the desired product based upon HPLC analysis).

300 MHz ¹H NMR (CDCl₃) δ 1.19 (m, 6H); 1.69 (2H); 1.79 (s, 1H); 2.03 (s,3H); 2.52 (m, 1H); 3.48 (m, 2H); 3.62 (m, 2H); 4.04 (m, 2H); 4.16 (m,2H); 4.61 (t, 1H); 5.12 (bs, 2H); 7.81 (s, 1H).

f) Preparation of

Into a 500 mL 1 neck round bottom flask was charged benzyl alcohol (136mL), cooled to 0° C., followed by portionwise addition of KO-t-Bu (36 g,321 mmol). The temperature was allowed to warm to 40° C., and themixture was stirred 20 minutes. To this mixture was added at 0° C. thecrude product of Example 14 step e) (24.7 g, 64.2 mmol) dissolved in 25mL anhydrous THF and benzyl alcohol (30 mL). The temperature was allowedto slowly warm to 8° C. over 2 hours. The reaction mixture was pouredinto 500 mL ice and was extracted with 500 mL MTBE. The organic layerwas washed with 250 mL of brine, dried over Na₂SO₄, filtered andconcentrated in vacuo to afford 193 g of a benzyl alcohol solution ofthe desired product. HPLC analysis indicated that the solution contained25.96 g of the desired product.

300 MHz ¹H NMR (CDCl₃) δ 1.22 (m, 6H); 1.55 (2H); 2.18 (m, 1H); 3.15 (m,1H); 3.40 (m, 1H); 3.51 (m, 2H); 3.70 (m, 2H); 4.25 (m, 2H); 4.63 (t,1H); 4.90 (bs, 2H); 5.25 (m, 1H); 5.58 (s, 2H); 7.35 (m, 3H); 7.51 (m,2H); 7.72 (s, 1H).

MS=(M+H)⁺=416 (CI).

g) Preparation of

Into a 100 mL 1 neck round bottom flask was charged the crude product ofExample 14 step f) (9.65 g of the benzyl alcohol solution, containing1.30 g, 3.13 mmol of the product of Example 14, step f) dissolved inabsolute EtOH (20 mL). To this was added 0.45 g of 10% Pd/C slurried in5 mL absolute EtOH. The reaction flask was evacuated and charged with H2three times with a balloon of H₂. The reaction flask was pressurizedwith 1 atm. H2 and the reaction mixture was stirred overnight. Thereaction mixture was filtered through a pad of diatomaceous earth toremove Pd/C. The volatiles were removed in vacuo. The residue was mixedwith 25 mL of isopropyl acetate and then concentrated in vacuo. Theresidue was diluted with EtOAc (10 mL), seeded with the desired product,heated to reflux and then CH₃CN (2 mL) and MTBE (35 ml) were added. Themixture was stirred for 30 minutes. The precipitate was filtered anddried to a constant weight of 600 mg of the desired product.

300 MHz ¹H NMR (d6-DMSO) δ 1.16 (m, 6H); 1.45 (m, 1H); 1.61 (m, 1H);2.16 (m, 1H); 3.45 (m, 2H); 3.40 (m, 1H); 3.62 (m, 2H); 4.02 (m, 2H);4.53 (t, 1H); 4.85 (t, 1H); 6.55 (bs, 11H); 7.75 (s, 1H). MS=(M+H)⁺=416(CI).

h) Preparation of

Into a 25 mL 1 neck round bottom flask was charged the product ofExample 14 step g) (0.650 g, 2.0 mmol), pyridine (4 mL) and CH₂Cl₂ (2mL), DMAP (10 mg). The mixture was cooled to −5° C. and stearoylchloride (790 mg, 2.6 mmol) dissolved in CH₂Cl₂ (0.5 mL) was added over5 minutes. The resulting mixture was stirred 16 hours at −5° C. AbsoluteEtOH (0.138 g, 3.0 mmol) was added and the mixture was stirred anadditional 1 hour. The reaction mixture was concentrated in vacuo.Toluene (30 mL) was added to the residue and then the mixture wasconcentrated in vacuo. Again, toluene (30 mL) was added to the residueand then the mixture was concentrated in vacuo. To the residue was added1% KH₂PO₄ (25 mL) and this mixture was extracted with CH₂Cl₂ (60 mL).The organic layer was separated and was dried over Na₂SO₄, filtered andconcentrated in vacuo to a constant weight of 1.65 g. The crude productwas chromatographed on 40 g of SiO₂, eluting with 95/5 CH₂Cl₂-EtOH,affording 367 mg of the desired product.

300 MHz ¹H NMR (CDCl₃) δ 0.89 (t, 3H); 1.26 (m, 30H); 1.65 (m, 3H); 2.32(m, 1H); 3.45 (m, 1H); 3.60 (m, 2H); 4.08 (m, 2H); 4.60 (m, 1H); 6.0(bs, 2H); 7.53 (s, 1H).

i) Preparation of

Into a 25 mL 1 neck round bottom flask was charged the product ofExample 14, step h) (0.234 g, 0.394 mmol) dissolved in THF (1.7 mL). Tothis solution was added triflic acid (0.108 g) in H₂O 180 mg. Themixture was stirred overnight at room temperature. To the reactionmixture was added saturated NaHCO₃ solution (10 mL), THF (5 mL), CH₂Cl₂(2 mL) and NaBH₄ (0.10 g). This mixture was stirred for 30 minutes. Tothe reaction mixture was added a 5% solution of KH₂PO₄ (30 mL). Thismixture was extracted with 2×15 ml of CH₂Cl₂. The organic layers werecombined and dried over Na₂SO₄, filtered and concentrated in vacuo to aconstant weight of 207 mg. This material was recrystallized from EtOAc(8 mL) and CH₃CN (0.5 mL) affording 173 mg of the desired product.

300 MHz ¹H NMR (d6-DMSO) δ 0.82 (t, 3H); 1.19 (m, 30H); 1.41 (m, 4H);2.19 (t, 2H); 2.32 (m, TH); 3.40 (m, 2H); 3.9 (m, 4H); 4.49 (m, 1H); 6.4(bs, 2H); 7.61 (m, 1.5H); 9.55 (m, 0.5H).

EXAMPLE 15 Alternative preparation of(R)-9-[4-(N-tert-butyloxycarbonyl-L-valyloxy)-2-(stearoyloxymethyl)butyl]guanine

(R)-9-[2-(Stearoyloxymethyl)-4-(t-butyldiphenylsilyloxy)butyl]guanine(45 g) and THF (950 ml) were combined in a 2 L flask. Then Boc-L-valine(3.22 g, 0.25 eq) was added, followed by tetrabutylammonium fluoride (1Min THF, 89.05 mL) over 10 minutes. The clear reaction mixture wasstirred at room temperature for 2 hours and 50 minutes with monitoringof the reaction progress by TLC (90/10 CH₂Cl₂/MeOH).

To the reaction mixture was added Boc-L-valine (35.43 g, 2.75 eq), DCC(36.67 g, 2.75 eq) and dimethylaminopyridine (1.1 g, 0.15 eq) in THF (25ml). The reaction mixture was stirred at room temperature for 24 hours.DCU was filtered off and washed with CH₂Cl₂. The filtrate wasconcentrated, and the residue was taken up in 2 liters of CH₂CL₂ andwashed with 2 L of ½ saturated sodium bicarbonate and brine solutions.On drying and evaporation, approximately 100 g of crude product wasobtained. The material was purified by silica chromatography (6000 ml ofsilica) using 3% MeOH/CH₂Cl₂ to 5% MeOH/CH₂Cl₂ to obtain 38.22 mg of thedesired product.

EXAMPLE 16 Alternative preparation of(R)-9-[2-(stearoyloxymethyl)-4-(L-valyloxy) butyl]guanine a)(R)-9-[2-Hydroxymethyl)-4-(t-butyldiphenylsilyloxymethyl)butyl]guanine

H2G (450.0 g, 1.78 mol) and N,N dimethylformamide (6.4 kg) were chargedinto a Bucchi evaporator and the mixture warmed to dissolve the solid.The solution was concentrated to dryness under vacuum at no more than90° C. The resulting powder was transferred to a 22 liter flask withstirrer, addition funnel and temperature probe. N,N-dimethylformamide(1.7 kg) was added followed by pyridine (3.53 kg). The resultingsuspension was cooled to −10° C. under nitrogen and stirred at −5±5° C.as t-butylchlorodiphenylsilane (684 g, 2.49 mol) was added dropwise. Theresulting mixture was stirred at −5±5° C. until the reaction wascomplete (as monitored by TLC (10:1 methylene chloride/methanol) andHPLC (4.6×250 mm Zorbax Rx C8 (5 micron); 60:40 acetonitrile-aq. NH₄OAC(0.05 M) at 1.5 ml/min; UV detection at 254 nm)). Water (16 kg) wasadded and the mixture was stirred for 30 minutes to precipitate theproduct, then the mixture was cooled to 0° C. for 30 minutes. The solidwas isolated by filtration and the product cake was washed with coldwater and sucked dry with air to provide the crude product as anoff-white solid. The crude solid was taken up in pyridine (3 kg) andconcentrated under vacuum at 60° C. to remove water. The dry solidresidue was slurried with methanol (10 kg) at 60° C. for 1-2 hours andfiltered while hot. The filtrate was concentrated under vacuum and thesolid residue was refluxed with isopropyl acetate (7 kg) for 30 minutes.The mixture was cooled to 20° C. and filtered. The filter cake was driedunder vacuum at 50° C. to provide the title compound as a white solid(555 g).

b) (R)-9-[2-(Stearoyloxymethyl)-4-(t-butyldiphenylsilyloxy)butyl]guanine

The product of Example 16, step a) (555 g, 1.113 mol) was charged to a50 liter Buchi evaporator. Pyridine (2.7 kg) was added dropwise todissolve the solid and the mixture was distilled to dryness under vacuumat 60° C. The residue was taken up in fresh pyridine (2.7 kg) andtransferred to a 22 liter flask with stirrer, addition funnel andtemperature probe. The solution was cooled to −5° C. under nitrogen. Asolution of stearoyl chloride (440 g, 1.45 mol) in methylene chloride(1.5 kg) was added so as to maintain a temperature below 0° C.4-(N,N-dimethylamino)pyridine (15 g, 0.12 mol) was added and the mixturewas stirred at −5-0° C. for 2-4 hours until conversion was complete (asmonitored by TLC (10:1 methylene chloride/methanol) and HPLC (4.6×250 mmZorbax Rx C8 (5 micron); 60:40 acetonitrile-aq. NH₄OAc (0.05 M) at 1.5ml/min; UV detection at 254 nm)). At the end of the reaction,acetonitrile (8.7 kg) was added and the mixture was stirred for not lessthan 15 minutes to precipitate the product. The slurry was cooled to 0°C. for 2 hours and the solid isolated by filtration and the filter cakewashed with acetonitrile (2 kg). The desired product was obtained as awhite solid (775 g).

c) (R)-9-[4-Hydroxy-2-(stearoyloxymethyl)butyl]guanine

A solution of the product of Example 16, step b) (765 g, 0.29 mol) intetrahydrofuran (10 kg) was prepared in a reactor. A solution oftetra(n-butyl)ammonium fluoride in tetrahydrofuran (1.7 kg of 1 Msolution, 1.7 mol) was added and the resulting clear solution wasstirred at 20±5° C. for 4 hours. Water (32 kg) was added and theresulting slurry was stirred for 1 hour and then cooled to 0° C. for 30minutes. The precipitate was isolated by filtration and the filter cakewas washed successively with water (10 kg) and acetonitrile (5 kg).After drying under vacuum at 25° C., 702 g of crude product wasobtained. The crude product was dissolved in refluxing THF (4.2 kg) andwater (160 g), then cooled to 40° C. and treated with methylene chloride(14.5 kg). The mixture was allowed to cool to 25±5° C. for 1 hour, thenit was cooled to 5±5° C. for 1 hour to complete precipitation. Theslightly off-white powder was isolated by filtration and dried undervacuum at 40° C. to yield the desired product (416 g).

d) (R)-9-[4-(N-Cbz-L-valyloxy)-2-(stearoyloxymethyl)butyl]guanine

A solution of N-Cbz-L-valine (169 g, 0.67 mol) in dry THF (750 ml) wasprepared in a 2 liter flask with mechanical stirrer, thermometer andaddition funnel. A solution of dicyclohexylcarbodiimide (69.3 g, 0.34mol) in THF (250 ml) was added over 5 minutes and the resulting slurrywas stirred at 20±5° C. for 2 hours. The slurry was filtered and thefilter cake was washed with THF (300 ml). The filtrate and wash werecharged to a 3 liter flask with stirrer and thermometer. The product ofExample 16, step c) (116 g, 0.22 mol) was added as a solid, with a rinseof THF (250 ml). 4-(N,N-dimethylamino)pyridine (2.73 g, 0.022 mol) wasadded and the white slurry stirred at 20±5° C. Within 15 minutes, thesolids were all dissolved and the reaction was complete within 1 hour(as determined by HPLC: 4.6×250 mm Zorbax Rx C8 column; 85:15acetonitrile-0.2% aq. HClO₄ at 1 ml/min.; UV detection at 254 nm;starting material elutes at 4.1 min. and product elutes at 5.9 min.).The reaction was quenched by addition of water (5 ml) and the solutionwas concentrated under vacuum to leave a light yellow semisolid. Thiswas taken up in methanol (1.5 liters) and warmed to reflux for 30minutes. The solution was cooled to 25° C. and the precipitate wasremoved by filtration. The filtrate was concentrated under vacuum toleave a viscous, pale yellow oil. Acetonitrile, (1 L) was added and theresulting white suspension was stirred at 20±5° C. for 90 minutes. Thecrude solid product was isolated by filtration, washed with acetonitrile(2×100 ml) and air-dried overnight to provide the desired product as awaxy, sticky solid (122 g). This was further purified by crystallizationfrom ethyl acetate (500 ml) and drying under vacuum at 30° C. to providethe desired product as a white, waxy solid (104 g).

e) (R)-9-[4-(L-valyloxy)-2-(stearoyloxymethyl)butyl]guanine

A solution of the product of Example 16, step d), (77 g) in warm (40°C.) ethanol (2.3 L) was charged to an hydrogenation reactor with 5% Pd—C(15.4 g). The mixture was agitated at 40° C. under 40 psi hydrogen for 4hours, evacuated and hydrogenated for an additional 4-10 hours. Thecatalyst was removed by filtration and the filtrate was concentratedunder vacuum to provide a white solid. This was stirred with ethanol(385 ml) at 25° C. for 1 hour, then cooled to 0° C. and filtered.

The filter cake was dried with air, then under vacuum at 35° C. to yieldthe title compound as a white powder (46 g).

EXAMPLE 17 (R)-9-[2-(L-Valyloxymethyl)-4-(stearoyloxy)butyl]guanine a)(R)-9-[2-Hydroxymethyl-4-(stearoyloxy)butyl]guanine

H2G (506 mg; 2.0 mmol) was dissolved in dry N,N-dimethylformamide (40ml) with pyridine (400 mg; 5.06 mmol) and 4-dimethylaminopyridine (60mg; 0.49 mmol). Stearoyl chloride (1500 mg; 4.95 mmol) was added and themixture kept overnight at room temperature. Most of the solvent wasevaporated in vacuo, the residue stirred with 70 ml ethyl acetate and 70ml water, and the solid filtered off, washed with ethyl acetate andwater and dried to yield 680 mg of crude product. Column chromatographyon silica gel (chloroform:methanol 15:1) gave pure title compound as awhite solid.

¹H NMR (DMSO-d₆) δ: 0.86 (t, 3H); 1.25 (s, 28H); 1.51 (qui, 2H); 1.62(m, 2H); 2.06 (m, 1H); 2.23 (t, 2H); 3.34 (d, 2H); 3.96 (ABX, 2H); 4.07(dd, 2H); 6.30 (br s, 2H); 7.62 (s, 1H); 10.45 (s, 1H).

¹³C NMR (DMSO-d₆) δ: 13.8 (C18); 22.0 (C17); 24.4 (C3); 27.7 (C3′);28.4-28.8 (C₄₋₆, C15); 28.9 (C₇₋₁₄); 31.2 (C16); 33.5 (C2); 38.0 (C2′);44.0 (C1′); 60.6/61.8 (C4′, C2″); 116.5 (guaC5); 137.7 (guaC7); 151.4(guaC4); 153.5 (guaC2); 156.7 (guaC6); 172.7 (COO).

b) (R)-9-[2-(N-Boc-L-valyloxymethyl)-4-(stearoyloxy)butyl]guanine

A mixture of N-Boc-L-valine (528 mg; 2.1 mmol) and N,N′-dicyclohexylcarbodiimide (250 mg; 1.21 mg) in dichloromethane (20 ml) was stirredover night at room temperature, dicyclohexylurea filtered off andextracted with a small volume of dichloromethane, and the filtrateevaporated in vacuo to a small volume.(R)-9-[2-Hydroxymethyl-4-(stearoyloxy)butyl]guanine (340 mg; 0.654mmol), 4-dimethylaminopyridine (25 mg; 0.205 mmol), and dryN,N-dimethylformamide (15 ml) were added and the mixture was stirred for4 h at 50° C. under N₂. The solvent was evaporated in vacuo to a smallvolume. Column chromatography on silica gel, then on aluminum oxide(ethyl acetate:methanol:water 15:2:1 as eluent) gave 185 mg (39%) puretitle compound as a white solid.

¹H NMR (CHCl₃) δ: 0.85-1.0 (m, 9H) 18-CH₃, CH(CH₃)₂; 1.25 (s, 28H)4-17-CH₂; 1.44 (s, 9H) t-Bu; 1.60 (qui, 2H) 3-CH₂; 1.74 (qua, 2H)3′-CH₂; 2.14 (m, 1H) 2′-CH; 2.29 (t, 2H) 2-CH₂; 2.41 (m, 1H)CH(CH₃)₂;4.1-4.3 (m, 6H)C1′-CH₂, C2″-CH₂, C4-CH₂; 5.4 (d, 1H) αCH; 6.6 (br s, 2H)guaNH₂; 7.73 (s, 1H) guaH8; 12.4 (br s).

¹³C NMR (CHCl₃) δ: 13.9 (C18); 17.5/18.9 (2 Val CH₃); 22.4 (C17); 24.7(C3); 28.1 (C3′); 28.9-29.3 (C₄₋₆, C15); 29.4 (C₇₋₁₄); 30.7 (Val αC);31.7 (C16); 34.0 (C2); 35.9 (C2′); 43.9 (C1′); 58.7 (Val αC); 61.4/63.6(C4′, C2″); 79.9 (CMe₃); 116.4 (guaC5); 137.9 (guaC7); 151.7 (guaC4);153.7 (guaC2); 155.7 (CONH); 158.8 (guaC6); 172.1 (CHCOO); 173.5(CH₂COO).

c) (R)-9-[2-(L-Valyloxymethyl)-4-(stearoyloxy)butyl]guanine

Chilled trifluoroacetic acid (2.0 g) was added to(R)-9-[2-(N-Boc-L-valyloxymethyl)-4-(stearoyloxy)butyl]guanine (180 mg;0.25 mmol) and the solution kept at room temperature for 1 h, evaporatedto a small volume, and lyophilized repeatedly with dioxane until a whiteamorphous powder was obtained. The yield of title compound, obtained asthe trifluoracetate salt, was quantitative.

¹H NMR (DMSO-d₆) δ: 0.87 (t, 3H) 18-CH₃, 0.98 (dd, 6H)CH(CH₃)₂; 1.25 (s,28H) 4-17-CH₂; 1.50 (qui, 2H) 3-CH₂; 1.68 (qua, 2H) 3′-CH₂; 2.19 (m, 1H)2′-CH; 2.26 (t, 2H) 2-CH₂; 2.40 (m, 1H)CH(CH₃)₂; 3.9-4.25 (m,7H)C1′-CH₂, C2″-CH₂, C4-CH₂, αCH; 6.5 (br s, 2H) guaNH₂; 7.79 (s, 1H)guaH8; 8.37 (br s, 3H) NH₃ ⁺; 10.73 (br s, 1H) guaNH.

¹³C NMR (DMSO-d₆) δ: 14.2 (C18); 17.9/18.3 (2 Val CH₃); 22.3 (C17); 24.6(C3); 27.7 (C3′); 28.7-29.1 (C4-6, C15); 29.2 (C₇₋₁₄); 29.5 (Val βC);31.5 (C16); 33.7 (C2); 35.0 (C2′); 44.1 (C1′); 57.6 (Val αC); 61.6/65.2(C4′, C2″); 116.1 (guaC5); 116.3 (qua, J 290 Hz, CF₃); 137.9 (guaC7);151.5 (guaC4); 154.0 (guaC2); 156.7 (guaC6); 158.3 (qua, J=15 Hz,CF₃COO) 169.1 (CHCOO); 173.1 (CH₂COO).

EXAMPLE 18 Alternative preparation of(R)-9-[2-hydroxymethyl-4-(stearoyloxybutyl]guanine

H2G (7.60 g, 30 mmol) was heated to solution in dry DMF (200 ml). Thesolution was filtered to remove solid impurities, cooled to 20° C. (H2Gcrystallized) and stirred at that temperature during addition ofpyridine (9.0 g, 114 mmol), 4-dimethylaminopyridine (0.46 g, 3.75 mmol)and then, slowly, stearoyl chloride (20.0 g, 66 mmol). Stirring wascontinued at room temperature overnight. Most of the solvent was thenevaporated off in vacuo, the residue stirred with 200 ml ethyl acetateand 200 ml water and the solid filtered off, washed with ethyl acetateand water and dried to yield crude product. As an alternative torecrystallization, the crude product was briefly heated to almostboiling with 100 ml of ethyl acetate:methanol:water (15:2:1) and thesuspension slowly cooled to 30° C. and filtered to leave most of the 2″isomer in solution (the 2″ isomer would crystallize at lowertemperature). The extraction procedure was repeated once more to yield,after drying in vacuo, 6.57 g (42%) of almost isomer free product.

EXAMPLE 19 Preparation of crystalline(R)-9-[2-stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine

The product of Example 16, step c) (20.07 g, 32.5 mmol) was dissolved inabsolute ethanol (400 ml) with heating, filtered, and further dilutedwith ethanol (117.5 ml).

To this solution was added water (HPLC grade, 103.5 ml), and the mixturewas allowed to cool to 35-40° C. After the mixture was cooled, water(HPLC grade, 931.5 ml) was added at a constant rate over 16 hours withefficient stirring. After all the water was added, stirring wascontinued for 4 hours at room temperature. The resulting precipitate wasfiltered through paper and dried under vacuum at room temperature toobtain the title compound as a white, free flowing crystalline powder(19.43 g, 97%), m pt 169-170° C.

EXAMPLE 20 9-R-(4-Hydroxy-2-(L-valyloxymethyl)butyl) guanine

a) To a solution of9-R-(4-(tert-butyldiphenylsilyloxy)-2-(hydroxymethyl)butyl)guanine (695mg, 1.5 mmole) in DMF (30 ml) were added N-Boc-L-Valine (488 mg, 2.25mmole), 4-dimethylamino pyridine (30 mg, 0.25 mmole) and DCC (556 mg,2.7 mmole). After 16 hr, the reaction was recharged with N-Boc-L-valine(244 mg) and DCC (278 mg), and was kept for an additional 5 hours. Thereaction mixture was filtered through Celite and poured into sodiumhydrogen carbonate aqueous solution, and then it was extracted withdichloromethane. The organic phase was evaporated and purified by silicagel column chromatography, giving 950 mg the N-protected monoamino acylintermediate.

b) The above intermediate (520 mg, 0.78 mmole) was dissolved in THF (15ml). To the solution was added hydrogen fluoride in pyridine (70%/30%,0.34 ml). After two days, the solution was evaporated and coevaporatedwith toluene. Purification by silica gel column chromatography gave 311mg of the protected monoamino acyl compound.

¹H-NMR (DMSO-d6): δ 10.41 (s, 1H), 7.59 (1H), 6.26 (br s, 2H), 4.32 (t,1H), 3.95 (m, 5H), 3.46 (m, 2H), 2.41 (m, 1H), 2.06 (m, 1H), 1.45 (m,2H), 1.39 (s, 9H), 0.90 (d, 6H).

c) The product of step b) (95 mg, 0.21 mmole) was treated with a mixtureof trifluoroacetic acid (4 ml) and dichloromethane (6 ml) for 1 hr. Thesolution was evaporated and freeze-dried, give 125 mg of the unprotectedmonoaminoacyl product.

¹H-NMR (D₂O): δ 8.88 (s, 1H), 4.32 (m, 4H), 3.96 (d, 1H), 3.68 (m, 2H),2.63 (m, 1H), 2.22 (m, 1H), 1.73 (m, 2H), 1.00 (m, 6H).

EXAMPLE 21 (R)-9-(2-Hydroxymethyl-4-(L-isoleucyloxy)butyl) guanine

a) To a solution of (R)-9-(2-hydroxymethyl-4-hydroxybutyl)guanine (2.53g, 10 mmole) in DMF (250 ml) were added N-Boc-L-isoleucine (2.77 g, 12mmole), 4-dimethylaminopyridine (61 mg, 0.6 mmole) and DCC (3.7 g, 18mmole). After reaction for 16 hr at 0° C., N-Boc-L-isoleucine (1.3 g)and DCC (1.8 g) were recharged, and the reaction was kept overnight atroom temperature. The reaction mixture was filtered through Celite andthe filtrate was evaporated and purified by silica gel columnchromatography, giving 1.25 g of the N-protected monoamino acylintermediate.

¹H-NMR (DMSO-d6): δ 10.56 (s, 1H), 7.62 (s, 1H), 6.43 (s, 2H), 4.75 (t,1H), 4.15-3.80 (m, 5H), 3.25 (m, 2H) 2.05 (m, 1H), 1.80-1-05 (m, 14H),0.88 (m, 6H).

b) The intermediate from step a) (100 mg, 0.21 mmole) was treated withtrifluoroacetic acid (3 m) and for 30 min at 0° C. The solution wasevaporated and freeze-dried, give the titled unprotected mono-aminoacylproduct in quantitative yield.

¹H-NMR (DMSO-d6+D₂O): δ 8.72 (s, 11H), 4.15 (m, 4H), 3.90 (d, 11H), 3.42(m, 2H), 2.09 (m, 1H), 1.83 (m, 1H), 1.61 (m, 2H), 1.15 (m, H), 0.77 (d,3H), 0.71 (t, 3H).

EXAMPLE 22 (R)-9-[2-Hydroxymethyl-4-(L-valyloxy)butyl]guanine

The product of Example 1, step a) was deprotected with trifluoroaaceticacid in the same manner as Example 1, step c)

¹H-NMR (250 MHz, DMSO-d₆): δ 1.04 (dd, 6H), 1.55-1.88 (m, 2H), 2.21 (m,2H), 3.48 (m, 2H), 4.00 (m, 1H), 4.13 (m, 2H), 4.34 (t, 2H), 6.9 (br s,2H), 8.21 (s, 1H), 8.5 (br s, 3H), 11.1 (br s, 11H).

EXAMPLE 23 (R)-9-[2-(L-Valyloxymethyl)-4-(valyloxy)butyl]guanine a)(R)-9-[4-(N-Boc-L-valyloxy)-2-(N-Boc-L-valyloxymethyl)butyl]guanine

Application of the technique described in Example 1, step a), but using2.7 eqs, 0.28 eqs, and 3.2 eqs of N-Boc-L-valine, DMAP, and DCC,respectively, resulted in the title compound.

¹H NMR (250 MHz, CHCl₃) δ: 0.95 (m, 12H), 1.42 (br s, 18H), 1.8 (m, 2H),2.14 (m, 2H), 2.47 (m, 1H), 4.0-4.4 (m, 8H), 6.5 (br s, 2H), 7.67 (s,1H).

b) (R)-9-[4-(L-Valyloxy)-2-(L-valyloxymethyl)butyl]guanine

The titled compound was obtained as the tris-trifluoroacetate salt fromthe intermediate of Example 20 step a) by deprotection in a manneranalogous to Example 1 step c).

¹H NMR (250 MHz, D₂O) δ: 1.0 (m, 12H), 1.89 (m, 2H), 2.29 (m, 2H), 2.62(m, 1H), 4.02 (dd, 2H), 4.38 (m, 6H), 4.89 (br s, ca. 10H), 8.98 (s,11H).

EXAMPLE 24 (R)-9-[4-hydroxy-2-(stearoyloxymethyl)butyl]guanine

The titled compound is prepared according to steps a) to c) of Example7.

¹H NMR (250 MHz, DMSO-d₆): δ 10.52 (s, 1H), 7.62 (s, 1H), 6.39 (s, 2H),4.50 (t, 1H), 3.93 (m, 4H), 3.42 (m, 2H), 2.45 (m, 1H), 2.23 (t, 2H),1.48 (m, 4H), 1.22 (s, 28H), 0.89 (t, 3H)

EXAMPLE 25 (R)-9-[2-Hydroxymethyl-4-(stearoyloxy)butyl]guanine

The titled compound is prepared by the procedure of Example 17, step a)

¹H NMR (DMSO-d₆) δ: 0.86 (t, 3H); 1.25 (s, 28H); 1.51 (qui, 2H); 1.62(m, 2H); 2.06 (m, 1H); 2.23 (t, 2H); 3.34 (d, 2H); 3.96 (ABX, 2H); 4.07(dd, 2H); 6.30 (br s, 2H); 7.62 (s, 1H); 10.45 (s, 1H).

EXAMPLE 26 Alternative Preparation of(R)-9-[2-stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine a)(R)-9-[4-N-benzyloxycarbonyl-L-valyloxy)-2-(hydroxymethyl)-butyl]guanine

Dry H2G (252 mg, 1 mmol), 4-dimethylaminopyridine (122 mg, 1 mmol) andN-Cbz-L-valine p-nitrophenyl ester (408 mg, 1.1 mmol) were dissolved indry dimethyl formamide (16 ml). After stirring at 23° C. for 30 hours,the organic solvent was removed and the residue carefullychromatographed (silica, 2%-7% methanol/methylene chloride) to affordthe desired product as a white solid (151 mg, 31%).

b)(R)-9-[4-N-benzyloxycarbonyl-L-valyloxy)-2-(stearoyloxymethyl)-butyl]guanine

A solution of stearoyl chloride (394 mg, 1.3 mmol) in dry methylenechloride (2 ml) was added slowly dropwise under nitrogen to a solutionof the product of step a) (243 mg, 1 mmol) and 4-dimethylaminopyridine(20 mg) in dry pyridine (5 ml) at −5° C. The reaction mixture wasstirred at that temperature for 12 hours. Methanol (5 ml) was added andthe reaction stirred for 1 hour. After removal of the solvent, theresidue was triturated with acetonitrile and chromatographed (silica,0-5% methanol/methylene chloride) to afford the desired product (542 mg,72%).

c) (R)-9-[2-stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine

The product of step b) (490 mg, 1 mmol) was dissolved in methanol (30ml) and 5% Pd/C (100 mg) added. A balloon filled with hydrogen wasplaced on top of the reaction vessel. After 6 hours at 23° C., TLCshowed the absence of starting material. The reaction mixture wasfiltered through a 0.45 micron nylon membrane to remove the catalyst andthe solvent was removed to afford the desired product as a white solid(350 mg, 99%) which was identical (spectral and analytical data) toExample 16.

EXAMPLE 27 Alternative Preparation of(R)-9-(4-hydroxy-2-(L-valyloxymethyl)butyl)guanine

(R)-9-(4-(L-valyloxy)-2-(L-valyloxymethyl) butyl)guanine from Example 23step b) (100 mg, 0,126 mmole) was dissolved in 0.1 N NaOH aqueoussolution (6.3 ml, 0.63 mmole) at room temperature. At intervals, analiquot was taken and neutralized with 0.5 N trifluoroacetic acid. Thealiquots were evaporated and analyzed by HPLC to monitor the progress ofthe reaction. After 4 hours, 0.5 N trifluoroacetic acid solution (1.26ml, 0.63 mmole) was added to the solution and the reaction mixture wasevaporated. The desired product was purified by HPLC, (YMC, 50×4.6 mm,gradient 0.1% TFA+0-50% 0.1% TFA in acetonitrile, in 20 minutes, UVdetection at 254 nm. Yield: 13.6%

¹H-NMR (D₂O): δ 8.81 (s, 1H), 4.36 (m, 4H), 4.01 (d, 1H), 3.74 (m, 2H),2.64 (m, 1H), 2.25 (m, 1H), 1.73 (m, 2H), 1.03 (dd, 6H).

EXAMPLE 28 Alternative preparation of(R)-9-(2-hydroxymethyl-4-(L-valyloxy)butyl)guanine

HPLC separation of the reaction solution from Example 27 gave the titledcompound in 29.2% yield.

¹H-NMR (DMSO-d₆): δ 8.38 (s, 3H), 8.26 (s, 1H), 6.83 (br s, 2H), 4.23(m, 2H), 4.06 (m, 2H), 3.91 (m, 1H), 3.40 (m, 2H), 2.19 (m, 2H),1.8-1.40 (m, 2H), 0.95 (dd, 6H).

EXAMPLE 29 (R)-9-[2-stearoyloxymethyl)-4-(L-valyloxy)]butylguaninemonohydrochloride

The product of Example 16, step d) (360 mg, 0.479 mmol) was dissolved ina mixture of methanol (10 ml) and ethyl acetate (10 ml). To the solutionwas added 10% Pd/C (100 mg) and 1N HCl (520 microliters). The reactionmixture was stirred at room temperature for 2 hours under 1 atm. H2. Thereaction mixture was filtered and the solvent evaporated from thefiltrate to provide the desired product as a crystalline solid (300 mg).

FORMULATION EXAMPLE A Tablet Formulation

The following ingredients are screened through a 0.15 mm sieve anddry-mixed

10 g (R)-9-[2-(stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine 40 glactose 49 g crystalline cellulose  1 g magnesium stearate

A tabletting machine is used to compress the mixture to tabletscontaining 250 mg of active ingredient.

FORMULATION EXAMPLE B Enteric Coated Tablet

The tablets of Formulation Example A are spray coated in a tablet coaterwith a solution comprising

120 g ethyl cellulose 30 g propylene glycol 10 g sorbitan monooleate ad1 000 ml aq. dist.

FORMULATION EXAMPLE C Controlled Release Formulation

50 g (R)-9-[2-(stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine 12 ghydroxypropylmethylcellulose (Methocell K15) 4.5 g  lactoseare dry-mixed and granulated with an aqueous paste of povidone.Magnesium stearate (0.5 g) is added and the mixture compressed in atabletting machine to 13 mm diameter tablets containing 500 mg activeagent.

FORMULATION EXAMPLE D Soft Capsules

250 g (R)-9-[2-(stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine 100 glecithin 100 g arachis oil

The compound of the invention is dispersed in the lecithin and arachisoil and filled into soft gelatin capsules.

BIOLOGY EXAMPLE 1 Bioavailability Testing in Rats

The bioavailability of compounds of the invention were compared to theparent compound H2G and other H2G derivatives in a rat model. Compoundsof the invention and comparative compounds were administered, per oral(by catheter into the stomach), to multiples of three individuallyweighed animals to give 0.1 mmol/kg of the dissolved prod rug in anaqueous (Example 4, 5, Comparative example 1-3, 5, 8), peanut oil(Comparative examples 4, 9, 10) or propylene glycol (Example 1-3, 6-12,17, Comparative example 6, 7) vehicle dependent on the solubility of thetest compound ingredient. The animals were fasted from 5 hours before toapproximately 17 hours after administration and were maintained inmetabolic cages. Urine was collected for the 24 hours followingadministration and frozen until analysis. H2G was analysed in the urineusing the HPLC/UV assay of Stahle & Öberg, Antimicrob Agents Chemother.36 No 2, 339-342 (1992), modified as follows: samples upon thawing arediluted 1:100 in aq dist H₂O and filtered through an amicon filter withcentrifugation at 3000 rpm for 10 minutes. Duplicate 30 μl samples arechromatographed on an HPLC column; Zorbax SB-C18; 75×4.6 mm; 3.5 micron;Mobile phase 0.05M NH₄PO₄, 3-4% methanol, pH 3.3-3.5; 0.5 ml/min; 254nm, retention time for H2G at MeOH 4% and pH 3.33, ˜12.5 min.Bioavailability is calculated as the measured H2G recovery from eachanimal averaged over at least three animals and expressed as apercentage of the averaged 24 hour urinary H2G recovery from a group of4 individually weighed rats respectively injected i.vjugularis with 0.1mmol/kg H2G in a Ringer's buffer vehicle and analysed as above.

Comparative example 1 (H2G) was from the same batch as used forpreparation of Examples 1 to 12. The preparation of Comparative example2 (monoVal-H2G) and 3 (diVal-H2G) are shown in Examples 21 and 23.Comparative example 4 (distearoyl H2G) was prepared by di-esterificationof unprotected H2G in comparable esterification conditions to step 2 ofExample 1. Comparative examples 5 & 8 (Val/Ac H2G) were preparedanalogously to Example 4 using acetic anhydride with relevant monovalineH2G. Comparative example 6 (Ala/stearoyl H2G) was prepared analogouslyto Example 6 using N-t-Boc-L-alanine in step 4. Comparative example 7(Gly/decanoyl) was prepared analogously to Example 5 but using the step1 intermediate made with N-t-Boc-L-glycine. The preparation ofComparative examples 9 and 10 is shown in Examples 24 and 25respectively. The results appear on Table 2 overleaf:

TABLE 2 Compound R₁ R₂ Bioavailability Comparative example 1 hydrogenhydrogen 8% Comparative example 2 valyl hydrogen 29% Comparative example3 valyl valyl 36% Example 1 valyl stearoyl 56% Comparative example 4stearoyl stearoyl 1% Example 2 valyl myristoyl 57% Example 3 valyloleoyl 51% Example 4 valyl butyryl 45% Comparative example 5 valylacetyl 11% Example 5 valyl decanoyl 48% Example 6 valyl docosanoyl 48%Example 7 isoleucyl stearoyl 53% Example 8 isoleucyl decanoyl 57%Example 9 isoleucyl myristoyl 49% Example 10 valyl 4-acetylbutyryl 52%Example 11 valyl dodecanoyl 46% Example 12 valyl palmitoyl 58% Example17 stearoyl valyl 52% Comparative example 6 alanyl stearoyl 23%Comparative example 7 glycyl decanoyl 25% Comparative Example 8 acetylvalyl 7% Comparative Example 9 hydrogen stearoyl 12% Comparative Example10 stearoyl hydrogen 7%

Comparison of the bioavailabilities of the compounds of the inventionwith the comparative examples indicates that the particular combinationof the fatty acids at R₁/R₂ with the amino acids at R₁/R₂ producesbioavailabilities significantly greater than the corresponding diaminoacid ester or difatty acid ester. For example, in this model, thecompound of Example 1 displays 55% better bioavailability than thecorresponding divaline ester of Comparative example 3. The compound ofExample 4 displays 25% better availability than the correspondingdivaline ester.

It is also apparent, for instance from Comparative examples 5, 6 and 7that only the specified fatty acids of this invention in combinationwith the specified amino acids produce these dramatic and unexpectedincreases in pharmacokinetic parameters.

BIOLOGY EXAMPLE 2 Plasma Concentrations in Rats

A plasma concentration assay was done in male Sprague Dawley derivedrats. The animals were fasted overnight prior to dosing but werepermitted free access to water. Each of the compounds evaluated wasprepared as a solution/suspension in propylene glycol at a concentrationcorresponding to 10 mg H2G/ml and shaken at room temperature for eighthours. Groups of rats (at least 4 rats in each group) received a 10mg/kg (1 ml/kg) oral dose of each of the compounds; the dose wasadministered by gavage. At selected time points after dosing (0.25, 0.5,1, 1.5, 2, 4, 6, 9, 12, 15, and 24 hours after dosing), heparinizedblood samples (0.4 ml/sample) were obtained from a tail vein of eachanimal. The blood samples were immediately chilled in an ice bath.Within two hours of collection, the plasma was separated from the redcells by centrifugation and frozen till analysis. The components ofinterest were separated from the plasma proteins using acetonitrileprecipitation. Following lyophilisation, and reconstitution, the plasmaconcentrations were determined by reverse phase HPLC with fluorescencedetection. The oral uptake of H2G and other test compounds wasdetermined by comparison of the H2G area under the curve derived fromthe oral dose compared to that obtained from a 10 mg/kg intravenous doseof H2G, administered to a separate group of rats. The results aredepicted in Table 1B above.

BIOLOGY EXAMPLE 3 Bioavailability in Monkeys

The compounds of Example 1 and Comparative example 3 (see BiologyExample 1 above) were administered p.o. by gavage to cynomolgus monkeys.The solutions comprised:

-   Example 1 150 mg dissolved in 6.0 ml propylene glycol, corresponding    to 25 mg/kg or 0.0295 mmol/kg.-   Comparative 164 mg dissolved in 7.0 ml water, corresponding to 23.4-   Example 3 mg/kg or 0.0295 mmol/kg.

Blood samples were taken at 30 min, 1, 2, 3, 4, 6, 10 and 24 hours.Plasma was separated by centrifugation at 2500 rpm and the samples wereinactivated at 54° C. for 20 minutes before being frozen pendinganalysis. Plasma H2G levels were monitored by the HPLC/UV assay ofExample 30 above.

FIG. 1 depicts the plasma H2G recovery as a function of time. Althoughit is not possible to draw statistically significant conclusions fromsingle animal trials, it appears that the animal receiving the compoundof the invention experienced a somewhat more rapid and somewhat greaterexposure to H2G than the animal which received an alternative prodrug ofH2G.

BIOLOGY EXAMPLE 4 Antiviral Activity

Herpes simplex virus-1 (HSV-1)-infected mouse serves as an animal modelto determine the efficacy of antiviral agents in vivo. Mice inoculatedintraperitoneally with HSV-1 at 1000 times the LD₅₀ were administeredeither with a formulation comprising the currently marketed anti-herpesagent acyclovir (21 and 83 mg/kg in a 2% propylene glycol in sterilewater vehicle, three times daily, p.o.) or the compound of Example 29(21 and 83 mg/kg in a 2% propylene glycol in sterile water vehicle,three times daily, p.o.) for 5 consecutive days beginning 5 hours afterinoculation. The animals were assessed daily for deaths. The results aredisplayed in FIG. 2 which charts the survival rate against time. In thelegend, the compound of the invention is denoted Ex. 29 and acyclovir isdenoted ACV. The percentage of mice surviving the HSV-1 infection wassignificantly greater following a given dose of the compound of theinvention relative to an equivalent dose of acyclovir.

The foregoing is merely illustrative of the invention and is notintended to limit the invention to the disclosures made herein.Variations and changes which are obvious to one skilled in the art areintended to be within the scope and nature of the invention as definedin the appended claims

The invention claimed is:
 1. A method for the treatment of a viralinfection caused by a retrovirus comprising administering an effectiveamount of a compound having the Formula I

where a) R₁ is —C(O)CH(CH(CH₃)₂)NH₂ or —C(O)CH(CH(CH₃)CH₂CH₃)NH₂ and R₂is —C(O)C₃-C₂₁ saturated or monounsaturated substituted alkyl,optionally substituted with up to five similar or different substituentsindependently selected from the group consisting of hydroxy, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆ alkoxy C₁-C₆ alkyl, C₁-C₆ alkanoyl, amino,halo, cyano, azido, oxo, mercapto and nitro; or b) R₁ is —C(O)C₃-C₂₁saturated or monounsaturated alkyl, optionally substituted with up tofive similar or different substituents independently selected from thegroup consisting of hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkoxyC₁-C₆ alkyl, C₁-C₆ alkanoyl, amino, halo, cyano, azido, oxo, mercaptoand nitro, and R₂ is —C(O)CH(CH(CH₃)₂)NH₂ or —C(O)CH(CH(CH₃)CH₂CH₃)NH₂;or a pharmaceutically acceptable salt thereof, to a human or animal inneed thereof.
 2. A method according to claim 1, wherein R₁ is—C(O)CH(CH(CH₃)₂)NH₂ or —C(O)CH(CH(CH₃)CH₂CH₃)NH₂ and R₂ is —C(O)C₃-C₂₁saturated or monounsaturated alkyl optionally substituted with up tofive similar or different substituents independently selected from thegroup consisting of hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkoxyC₁-C₆ alkyl, C₁-C₆ alkanoyl, amino, halo, cyano, azido, oxo, mercaptoand nitro.
 3. A method according to claim 1 wherein R₁ or R₂ is a—C(═O)C₉ to C₁₇, saturated or N:9 monounsaturated, alkyl.
 4. A methodaccording to claim 1, wherein the compound is selected from the groupconsisting of (R)-9-[2-(Stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine,(R)-9-[2-(Myristoyloxymethyl)-4-(L-valyloxy)butyl]guanine,(R)-9-[2-(Oleoyloxymethyl)-4-(L-valyloxy)butyl]guanine,(R)-9-[2-(Butyloxymethyl)-4-(L-valyloxy)butyl]guanine,(R)-9-[2-(Decanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,(R)-9-[2-(Docosanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,(R)-9-[4-(L-Isoleucyloxy)-2-(stearoyloxymethyl)butyl]guanine,(R)-9-[2-(Decanoyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,R)-9-[4-(L-Isoleucyloxy)-2-(myristoyloxymethyl)butyl]guanine,(R)-9-[2-(4-Acetylbutyryloxymethyl-4-(L-valyloxy)butyl]guanine,(R)-9-[2-Dodecanoyloxymethyl-4-(L-valyloxy)butyl]guanine,(R)-9-[2-Palmitoyloxymethyl-4-(L-valyloxy)butyl]guanine,(R)-9-[2-(L-Valyloxymethyl)-4-(stearoyloxy)butyl]guanine or apharmaceutically acceptable salt thereof.
 5. A method according to claim1, wherein the compound is denoted(R)-9-[2-(stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine or apharmaceutically acceptable salt thereof.
 6. The method according toclaim 1, wherein said retrovirus is selected from the group consistingof HIV-1, HIV-2 and SIV.
 7. A method according to claim 6, wherein thecompound is denoted(R)-9-[2-(stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine or apharmaceutically acceptable salt thereof.